Macrophage derived chemokine (MDC), MDC analogs, MDC inhibitor substances and uses thereof

ABSTRACT

The present invention provides purified and isolated polynucleotide sequences encoding a novel macrophage-derived C-C chemokine designated “Macrophage Derived Chemokine” (MDC), and polypeptide fragments and analogs thereof. Also provided are materials and methods for the recombinant or synthetic production of the chemokine, fragments, and analogs; and purified and isolated chemokine protein, and polypeptide fragments and analogs thereof. Also provided are antibodies reactive with the chemokine and methods of making and using all of the foregoing. Also provided are assays for identifying modulators of MDC chemokine activity.

This application is a continuation-in-part of U.S. patent applicationSer. No. 09/067,447, filed Apr. 28, 1998, and a continuation-in-part ofU.S. patent application Ser. No. 08/939,107, filed Sep. 26, 1997,(Attorney docket No. 27866/34188), and a continuation-in-part of U.S.patent application Ser. No. 08/660,542, filed Jun. 7, 1996, and acontinuation-in-part of U.S. patent application Ser. No. 08/558,658,filed Nov. 16, 1995, and a continuation-in-part of U.S. patentapplication Ser. No. 08/479,620, filed Jun. 7, 1995. These applicationsare hereby incorporated by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates generally to chemokines and moreparticularly to purified and isolated polynucleotides encoding a novelhuman C-C chemokine, to purified and isolated chemokine protein encodedby the polynucleotides, to chemokine analogs, to materials and methodsfor the recombinant production of the novel chemokine protein andanalogs, to antibodies reactive with the novel chemokine, to chemokineinhibitors, and to uses of all of the foregoing materials. Of particularinterest is the use of chemokine inhibitor substances to treat allergicconditions such as asthma.

BACKGROUND

Chemokines, also known as “intercrines” and “SIS cytokines”, comprise afamily of small secreted proteins (e.g., 70-100 amino acids and about8-10 kiloDaltons) which attract and activate leukocytes and thereby aidin the stimulation and regulation of the immune system. The name“chemokine” is derived from chemotactic cytokine, and refers to theability of these proteins to stimulate chemotaxis of leukocytes. Indeed,chemokines may comprise the main attractants for inflammatory cells intopathological tissues. See generally, Baggiolini et al., Annu. Rev.Immunol, 15: 675-705 (1997); and Baggiolini et al., Advances inImmunology, 55:97-179 (1994), both of which are incorporated byreference herein. While leukocytes comprise a rich source of chemokines,several chemokines are expressed in a multitude of tissues. Baggioliniet al. (1994), Table II.

Previously identified chemokines generally exhibit 20-70% amino acididentity to each other and contain four highly-conserved cysteineresidues. Based on the relative position of the first two of thesecysteine residues, chemokines have been further classified into twosubfamilies. In the “C-X-C” or “α” subfamily, encoded by genes localizedto human chromosome 4, the first two cysteines are separated by oneamino acid. In the “C-C” or “β” subfamily, encoded by genes on humanchromosome 17, the first two cysteines are adjacent. X-raycrystallography and NMR studies of several chemokines have indicatedthat, in each family, the first and third cysteines form a firstdisulfide bridge, and the second and fourth cysteines form a seconddisulfide bridge, strongly influencing the native conformation of theproteins. In humans alone, more than ten distinct sequences have beendescribed for each chemokine subfamily. Chemokines of both subfamilieshave characteristic leader sequences of twenty to twenty-five aminoacids.

The C-X-C chemokines, which include IL-8, GROα/β/γ, platelet basicprotein Platelet Factor 4 (PF4), IP-10, NAP2, and others, shareapproximately 25% to 60% identity when any two amino acid sequences arecompared (except for the GROα/β/γ members, which are 84-88% identicalwith each other). Most of the C-X-C chemokines (excluding IP-10 andPlatelet Factor 4) share a common E-L-R tri-peptide motif upstream ofthe first two cysteine residues, and are potent stimulants ofneutrophils, causing rapid shape change, chemotaxis, respiratory burstand degranulation. These effects are mediated byseven-transmembrane-domain rhodopsin-like G protein-coupled receptors; areceptor specific for IL-8 has been cloned by Holmes et al. Science,253:1278-80 (1991), while a similar receptor (77% identity) whichrecognizes IL-8, GRO and NAP2 has been cloned by Murphy and Tiffany,Science, 253:1280-83 (1991 Progressive truncation of the N-terminalamino acid sequence of certain C-X-C chemokines, including IL-8, isassociated with marked increases in activity.

The C-C chemokines, which include Macrophage Inflammatory ProteinsMIP-1αand MIP-1β, Monocyte chemoattractant proteins 1, 2, 3, and 4(MCP-1/2/3/4), RANTES, I-30 eotaxin, TARC, and others, share 25% to 70%amino acid identity with each other. Previously-identified C-Cchemokines activate monocytes, causing calcium flux and chemotaxis. Moreselective effects are seen on lymphocytes, for example, T lymphocytes,which respond best to RANTES. Several seven-transmembrane-domain Gprotein-coupled receptors for C-C chemokines have been cloned to date,including a C-C chemokine receptor-1 (CCR1) which recognizes, e.g.,MIP-1α and RANTES (Neote et al., Cell, 72:415425 (1993)); a CCR2receptor which has two splice variants and which recognizes, e.g., MCP-1(Charo el al., Proc. Nat. Acad. Sci., 91:2752-56 (1994)); CCR3, whichrecognizes, e.g., eotaxin, RANTES, and MCP-3 (Combadiere, J. Biol.Chem., 270:16491 (1995)); CCR4, which recognizes MIP-1α, RANTES, andMCP-1 (Power et al., J. Biol. Chem., 270:19495 (1995)); and CCR5, whichrecognizes MIP-1α, MIP-1β, and RANTES (Samson et al., Biochemstry,35:3362 (1996)). Several CC chemokines have been shown to act asattractants for activated T lymphocytes. See Baggiolini et al. (1997).

Truncation of the N-terminal amino acid sequence of certain C-Cchemokines also has been associated with alterations in activity. Forexample, mature RANTES (1-68) is processed by CD26 (a dipeptidylaminopeptidase specific for the sequence NH₂—X-Pro- . . . ) to generatea RANTES (3-68) form that is capable of interacting with and transducinga signal through CCR5 (like the RANTES (1-68) form), but is onehundred-fold reduced in its capacity to stimulate through the receptorCCR1. See Proost et al., J. Biol. Chem., 273(13): 7222-7227 (1998); andOravecz et al., J. Exp. Med, 186: 1865-1872 (1997). U.S. Pat. Nos.5,459,128, 5,705,360, and 5,739,103 to Rollins and Zhang purport todescribe N-terminal deletions of chemokine MCP-1 that inhibit receptorbinding to the corresponding endogenous chemokine.

The roles of a number of chemokines, particularly IL-8, have been welldocumented in various pathological conditions. See generally Baggioliniet al. (1994), supra, Table VII. Psoriasis, for example, has been linkedto over-production of IL-8, and several studies have observed highlevels of IL-8 in the synovial fluid of inflamed joints of patientssuffering from rheumatic diseases, osteoarthritis, and gout.

The role of C-C chemokines in pathological conditions also has beendocumented, albeit less comprehensively than the role of IL-8. Forexample, the concentration of MCP-1 is higher in the synovial fluid ofpatients suffering from rheumatoid arthritis than that of patientssuffering from other arthritic diseases. The MCP-1 dependent influx ofmononuclear phagocytes may be an important event in the development ofidiopathic pulmonary fibrosis. The role of C-C chemokines in therecruitment of monocytes into atherosclerotic areas is currently ofintense interest, with enhanced MCP-1 expression having been detected inmacrophage-rich arterial wall areas but not in normal arterial tissue.Expression of MCP-1 in malignant cells has been shown to suppress theability of such cells to form tumors in vivo. (See U.S. Pat. No.5,179,078, incorporated herein by reference.) A need therefore existsfor the identification and characterization of additional C-Cchemokines, to further elucidate the role of this important family ofmolecules in pathological conditions, and to develop improved treatmentsfor such conditions utilizing chemokine-derived products.

With respect to the involvement of chemokines in allergic diseases,interest has focused on chemokines belonging to the CC family, such asRANTES, eotaxin, eotaxin-2, MCP-3 and MCP-4, because of their ability tocause migration of human eosinophils in vitro and in viv. The ability ofthese chemokines to selectively activate human eosinophil migrationappears to be due primarily to their activation of chemokine receptorCCR3. A need exists to elucidate the involvement of these and otherchemokines in eosinophil stimulation and activation, to facilitatebetter treatments for late-phase allergic reactions, such as asthma [seeAalbers et al., Eur. Respir. J, 6:840(1993); and Frigas et al., J.Allergy Clint Immunol., 77:527(1986)], in which eosinophil activationand migration have been implicated.

Chemokines of the C-C subfamily have been shown to possess utility inmedical imaging, e.g., for imaging sites of infection, inflammation, andother sites having C-C chemokine receptor molecules. See, e.g., Kunkelet al., U.S. Pat. No. 5,413,778, incorporated herein by reference. Suchmethods involve chemical attachment of a labeling agent (e.g., aradioactive isotope) to the C-C chemokine using art recognizedtechniques (see, e.g., U.S. Pat. Nos. 4,965,392 and 5,037,630,incorporated herein by reference), administration of the labeledchemokine to a subject in a pharmaceutically acceptable carrier,allowing the labeled chemokine to accumulate at a target site, andimaging the labeled chemokine in vivo at the target site. A need in theart exists for additional new C-C chemokines to increase the availablearsenal of medical imaging tools.

The C-C chemokines RANTES, MIP-α, and MIP-1β also have been shown to bethe primary mediators of the suppressive effect of human T cells on thehuman immunodeficiency virus (HIV), the agent responsible for causinghuman Acquired Immune Deficiency Syndrome (AIDS). These chemokines showa dose-dependent ability to inhibit specific strains of HIV frominfecting cultured T cell lines [Cocchi et al., Science, 270:1811(1995)]. In addition, International patent publication number WO97/44462, filed by Institut Pasteur, describes the use of fragments andanalogs of the chemokine RANTES as antagonists, to block RANTESinteraction with its receptors, for the purpose of suppressing HIV. TheC-X-C chemokine stromal derived factor-1 (SDF-1) also is capable ofblocking infection by T-tropic HIV-1 strains. See Winkler et al.,Science, 279:389-393 (1998). However, the processes through whichchemokines exert their protective effects have not been fullyelucidated, and these chemokines in fact may stimulate HIV replicationin cells exposed to the chemokines before HIV infection. See Kelly etal., J. Immunol., 160:3091-3095 (1998). Moreover, not all tested strainsof the virus are equally susceptible to the inhibitory effects ofchemokines; therefore, a need exists for additional C-C chemokines foruse as inhibitors of strains of HIV.

Similarly, it has been established that certain chemokine receptors suchas CCR5 [international Patent Publication No. WO 97/44055, published 27Nov. 1997], CCR8, CCR2, and CXCR4) are essential co-receptors (with theCD4 receptor) for HIV-1 entry into susceptible cells, and thatprogression to AIDS is delayed in patients having certain variantalleles of these receptors. A need exists for additional therapeutics toinhibit HIV-1 infection and/or proliferation by interfering with HIV-1entry and/or proliferation in susceptible cells.

More generally, due to the importance of chemokines as mediators ofchemotaxis and inflammation, a need exists for the identification andisolation of new members of the chemokine family to facilitatemodulation of inflammatory and immune responses.

For example, substances that promote inflammation may promote thehealing of wounds or the speed of recovery from conditions such aspneumonia, where inflammation is important to eradication of infection.Modulation of inflammation is similarly important in pathologicalconditions manifested by inflammation. Crohn's disease, manifested bychronic inflammation of all layers of the bowel, pain, and diarrhea, isone such pathological condition. The failure rate of drug therapy forCrohn's disease is relatively high, and the disease is often recurrenteven in patients receiving surgical intervention. The identification,isolation, and characterization of novel chemokines facilitatesmodulation of inflammation.

Similarly, substances that induce an immune response may promotepalliation or healing of any number of pathological conditions. Due tothe important role of leukocytes (e.g., neutrophils and monocytes) incell-mediated immune responses, and due to the established role ofchemokines in leukocyte chemotaxis, a need exists for the identificationand isolation of new chemokines to facilitate modulation of immuneresponses.

Additionally, the established correlation between chemokine expressionand inflammatory conditions and disease states provides diagnostic andprognostic indications for the use of chemokines, as well as forantibody substances that are specifically immunoreactive withchemokines; a need exists for the identification and isolation of newchemokines to facilitate such diagnostic and prognostic indications.

In addition to their ability to attract and activate leukocytes, somechemokines, such as IL-8, have been shown to be capable of affecting theproliferation of non-leukocytic cells See Tuschil, J. Invest. Dermatol.,99:294-298 (1992). A need exists for the identification and isolation ofnew chemokines to facilitate modulation of such cell proliferation.

It will also be apparent from the foregoing discussion of chemokineactivities that a need exists for modulators of chemokine activities, toinhibit the effects of endogenously-produced chemokines and/or topromote the activities of endogenously-produced or exogenouslyadministered chemokines. Such modulators typically include smallmolecules, peptides chemokine fragments and analogs, and/or antibodysubstances. Chemokine inhibitors interfere with chemokine signaltransduction, i.e., by binding chemokine molecules, by competitively ornon-competitively binding chemokine receptors, and/or by interferingwith signal transduction downstream from the chemokine receptors. A needexists in the art for effective assays to rapidly screen putativechemokine modulators for modulating activity.

For all of the aforementioned reasons, a need exists for recombinantmethods of production of newly discovered chemokines, which methodsfacilitate clinical applications involving the chemokines and chemokineinhibitors.

SUMMARY OF THE INVENTION

The present invention provides novel purified and isolatedpolynucleotides and polypeptides, antibodies, and methods and assaysthat fulfill one or more of the needs outlined above.

For example, the invention provides purified and isolatedpolynucleotides (i.e., DNA and RNA, both sense and antisense strands)encoding a novel human chemokine of the C-C subfamily, herein designated“Macrophage Derived Chemokine” or “MDC”. Preferred DNA sequences of theinvention include genomic and cDNA sequences and chemically synthesizedDNA sequences. The cDNA and deduced amino acid sequence of human MDC hasbeen published. See, e.g., International Patent Publication No. WO96/40923, published 19 December 1996; and Godiska et al., J. Exp. Med,185(9): 1595-1604 (1997). Compare International Publication No. WO96/39521 (12 Dec. 1996); and Chang et al., J. Biol. Chem., 272(40):25229-25237 (1997).

Polynucleotides encoding non-human vertebrate forms of MDC, especiallymammalian and avian forms of MDC, also are intended as aspects of theinvention.

The nucleotide sequence of a cDNA, designated MDC cDNA, encoding thischemokine, is set forth in SEQ ID NO: 1, which sequence includes 5′ and3′ non-coding sequences. A preferred DNA of the present inventioncomprises nucleotides 20 to 298 of SEQ ID NO. 1, which nucleotidescomprise the MDC coding sequence.

The human MDC protein comprises a putative twenty-four amino acid signalsequence at its amino terminus. Another preferred DNA of the presentinvention comprises nucleotides 92 to 298 of SEQ ID NO. 1, whichnucleotides comprise the putative coding sequence of the mature(secreted) MDC protein, without the signal sequence.

The amino acid sequence of human chemokine MDC is set forth in SEQ IDNO: 2. Preferred polynucleotides of the present invention include, inaddition to those polynucleotides described above, polynucleotides thatencode the amino acid sequence set forth in SEQ ID NO: 2, and thatdiffer from the polynucleotides described in the preceding paragraphsonly due to the well-known degeneracy of the genetic code.

Similarly, since twenty-four amino acids (positions −24 to −1) of SEQ IDNO: 2 comprise a putative signal peptide that is cleaved to yield themature MDC chemokine, preferred polynucleotides include those whichencode amino acids 1 to 69 of SEQ ID NO: 2. Thus, a preferredpolynucleotide is a purified polynucleotide encoding a polypeptidehaving an amino acid sequence comprising amino acids 1-69 of SEQ ID NO:2.

Among the uses for the polynucleotides of the present invention is theuse as a hybridization probe, to identify and isolate genomic DNAencoding human MDC, which gene is likely to have a three exon/two intronstructure characteristic of C-C chemokines genes. (See Baggiolini et al.(1994), supra); to identify and isolate DNAs having sequences encodingnon-human proteins homologous to MDC; to identify human and non-humanchemokine genes having similarity to the MDC gene; and to identify thosecells which express MDC and the conditions under which this protein isexpressed. Polynucleotides encoding human MDC have been employed tosuccessfully isolate polynucleotides encoding at least three exemplarynon-human embodiments of MDC (rat, mouse, macaque). (See SEQ ID NOs:35-38 & 45-46.)

Hybridization probes of the invention also have diagnostic utility,e.g., for screening for inflammation in human tissue, such as colontissue. More particularly, hybridization studies using an MDCpolynucleotide hybridization probe distinguished colon tissue ofpatients with Crohn's disease (MDC hybridization detected in epithelium,lamina propria, Payer's patches and smooth muscle) from normalhuman-colon tissue (no hybridization above background).

Generally speaking, a continuous portion of the MDC cDNA of theinvention that is at least about 14 nucleotides, and preferably about 18nucleotides, is useful as a hybridization probe of the invention. Thus,in one embodiment, the invention includes a DNA comprising a continuousportion of the nucleotide sequence of SEQ ID NO: 1 or of the non-codingstrand complementary thereto, the continuous portion comprising at least18 nucleotides, the DNA being capable of hybridizing under stringentconditions to a coding or non-coding strand of a human MDC gene. Fordiagnostic utilities, hybridization probes of the invention preferablyshow hybridization specificity for MDC gene sequences. Thus, in apreferred embodiment hybridization probe DNAs of the invention fail tohybridize under the stringent conditions to other human chemokine genes(e.g., MCP-1 genes, MCP-2 genes, MCP-3 genes, RANTES genes, MIP-1αgenes, MIP-1β genes, and I-309 genes, etc.).

In another aspect, the invention provides a purified polynucleotidewhich hybridizes under stringent conditions to the non-coding strand ofthe DNA of SEQ ID NO: 1. Similarly, the invention provides a purifiedpolynucleotide which, but for the redundancy of the genetic code, wouldhybridize under stringent conditions to the non-coding strand of the DNAof SEQ ID NO: 1. Exemplary stringent hybridization conditions are asfollows: hybridization at 42° C. in 5×SSC, 20 mM NaPO₄, pH 6.8, 50%formamide; and washing at 42° C. in 0.2×SSC. Those skilled in the artunderstand that it is desirable to vary these conditions empiricallybased on the length and the GC nucleotide base content of the sequencesto by hybridized, and that formulas for determining such variationexist. [See, e.g., Sambrook et al., Molecular Cloning: a LaboratoryManual. Second Edition, Cold Spring Harbor, N.Y.: Cold Spring HarborLaboratory (1989).]

In another aspect, the invention includes plasmid and viral DNA vectorsincorporating DNAs of the invention, including any of the DNAs describedabove or elsewhere herein. Preferred vectors include expression vectorsin which the incorporated MDC-encoding cDNA is operatively linked to anendogenous or heterologous expression control sequence. Such expressionvectors may further include polypeptide-encoding DNA sequences operablylinked to the MDC-encoding DNA sequences, which vectors may be expressedto yield a fusion protein comprising the MDC polypeptide of interest.

In another aspect, the invention includes a prokaryotic or eukaryotichost cell stably transfected or transformed with a DNA or vector of thepresent invention. In preferred host cells, the mature MDC polypeptideencoded by the DNA or vector of the invention is expressed. The DNAs,vectors, and host cells of the present invention are useful, e.g., inmethods for the recombinant production of large quantities of MDCpolypeptides of the present invention. Such methods are themselvesaspects of the invention. For example, the invention includes a methodfor producing MDC wherein a host cell of the invention is grown in asuitable nutrient medium and MDC protein is isolated from the cell orthe medium.

Knowledge of DNA sequences encoding MDC makes possible determination ofthe chromosomal location of MDC coding sequences, as well asidentification and isolation by DNA/DNA hybridization of genomic DNAsequences encoding the MDC expression control regulatory sequences suchas promoters, operators, and the like.

According to another aspect of the invention, host cells may be modifiedby activating an endogenous MDC gene that is not normally expressed inthe host cells or that is expressed at a lower level than is desired.Such host cells are modified (e.g., by homologous recombination) toexpress MDC by replacing, in whole or in part, the naturally-occurringMDC promoter with part or all of a heterologous promoter so that thehost cells express MDC. In such host cells, the heterologous promoterDNA is operatively linked to the MDC coding sequences, i.e., controlstranscription of the MDC coding sequences. See, for example, PCTInternational Publication No. WO 94/12650; PCT International PublicationNo. WO 92/20808; and PCT International Publication No. WO 91/09955. Theinvention also contemplates that, in addition to heterologous promoterDNA, amplifiable marker DNA (e.g., ada, dhfr, and the multi-functionalCAD gene which encodes carbamyl phosphate synthase, aspartatetranscarbamylase, and dihydro-orotase) and/or intron DNA may berecombined along with the heterologous promoter DNA into the host cells.If linked to the MDC coding sequences, amplification of the marker DNAby standard selection methods results in co-amplification of the MDCcoding sequences in such host cells.

The DNA sequence information provided by the present invention alsomakes possible the development, by homologous recombination or“knockout” strategies [see, Capecchi, Science, 244: 1288-1292 (1989)],of rodents that fail to express functional MDC or that express a variantof MDC. Such rodents are useful as models for studying the activities ofMDC, MDC variants, and MDC modulators in vivo. Rodents having ahumanized immune system are useful as models for studying the activitiesof MDC and MDC modulators toward HIV infection and proliferation.

In yet another aspect, the invention includes purified and isolated MDCpolypeptides. Mammalian and avian MDC polypeptides are specificallycontemplated. A preferred peptide is a purified chemokine polypeptidehaving an amino acid sequence comprising amino acids 1 to 69 of SEQ IDNO: 2 (human mature MDC). Throughout the application, human mature MDCusually will be referred to simply as “MDC” or as “mature MDC”. Ininstances where context warrants, such as certain descriptions ofexperiments that involve both human and non-human mature MDCs and/orthat involve MDC fragments and analogs, human mature MDC will sometimesbe specifically referred to as “human” and will sometimes be referred toas “MDC(1-69).”

Mouse and Rat MDC polypeptides of the invention are taught in SEQ IDNOs: 36 and 38. The sequence in SEQ ID NO: 36 depicts a complete murineMDC, consisting of a 24 residue leader peptide (residues −24 to −1 ofSEQ ID NO: 36) and a 68 residue murine mature MDC. The sequence in SEQID NO: 38 depicts a partial rat MDC, consisting of 13 residues of theleader peptide (residues −13 to −1) and the complete 68 residue ratmature MDC.

The polypeptides of the present invention may be purified from naturalsources, but are preferably produced by recombinant procedures, usingthe DNAs, vectors, and/or host cells of the present invention, or arechemically synthesized. Purified polypeptides of the invention may beglycosylated or non-glyclosylated, water soluble or insoluble, oxidized,reduced, etc., depending on the host cell selected, recombinantproduction method, isolation method, processing, storage buffer, and thelike.

Moreover, an aspect of the invention includes MDC polypeptide analogswherein one or more amino acid residues is added, deleted, or replacedfrom the MDC polypeptides of the present invention, which analogs retainone or more of the biological activities characteristic of the C-Cchemokines, especially of MDC. The small size of MDC facilitateschemical synthesis of such polypeptide analogs, which may be screenedfor MDC biological activities (e.g., the ability to induce macrophagechemotaxis, or inhibit monocyte chemotaxis) using the many activityassays described herein. Alternatively, such polypeptide analogs may beproduced recombinantly using well-known procedures, such assite-directed mutagenesis of MDC-encoding DNAs of the invention,followed by recombinant expression of the resultant DNAs.

In a related aspect, the invention includes polypeptide analogs whereinone or more amino acid residues is added, deleted, or replaced from theMDC polypeptides of the present invention, which analogs lack thebiological activities of C-C chemokines or MDC, but which are capable ofcompetitively or non-competitively inhibiting the binding of MDCpolypeptides with a C-C chemokine receptor. Such polypeptides areuseful, e.g., for modulating the biological activity of endogenous MDCin a host, as well as useful for medical imaging methods describedabove.

Certain specific analogs of MDC are contemplated to modulate thestructure, intermolecular binding characteristics, and biologicalactivities of MDC. For example, amino-terminal (N-terminal) andcarboxy-terminal (C-terminal) deletion analogs (truncations) arespecifically contemplated to change MDC structure and function. Amongthe amino terminal deletion analogs that are specifically contemplatedare analogs wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino terminalresidues have been deleted (i.e., deletions up to the conserved cysteinepair at positions 12 and 13 of human, murine, and rat mature MDC). Asset forth in detail below, experimental data indicates that most or allof these analogs will possess reduced MDC biological activities and, infact, will act as inhibitors of one or more biological activities ofmature MDC.

Additionally, the following single-amino acid alterations (alone or incombination) are specifically contemplated: (1) substitution of anon-basic amino acid for the basic arginine and/or lysine amino acids atpositions 24 and 27, respectively, of SEQ ID NO: 2; (2) substitution ofa charged or polar amino acid (e.g., serine, lysine, arginine,histidine, aspartate, glutamate, asparagine, glutamine or cysteine) forthe tyrosine amino acid at position 30 of SEQ ID NO: 2, the tryptophanamino acid at position 59 of SEQ ID NO: 2, and/or the valine amino acidat position 60 of SEQ ID NO: 2; and (3) substitution of a basic orsmall, non-charged amino acid (e.g., lysine, arginine, histidine,glycine, alanine) for the glutamic acid amino acid at position 50 of SEQID NO: 2. Specific analogs having these amino acid alterations areencompassed by the following formula (SEQ ID NO: 25): Met Ala Arg LeuGln Thr Ala Leu Leu Val Val Leu Val Leu Leu Ala−24             −20                 −15                 −10 Val Ala LeuGln Ala Thr Glu Ala Gly Pro Tyr Gly Ala Asn Met Glu             −5                   1               5 Asp Ser Val Cys CysArg Asp Tyr Val Arg Tyr Arg Leu Pro Leu Xaa     10                  15                  20 Val Val Xaa His Phe XaaTrp Thr Ser Asp Ser Cys Pro Arg Pro Gly 25                  30                  35                  40 Val ValLeu Leu Thr Phe Arg Asp Lys Xaa Ile Cys Ala Asp Pro Arg                 45                  50                  55 Val Pro XaaXaa Lys Met Ile Leu Asn Lys Leu Ser Gln             60                  65wherein the amino acid at position 24 is selected from the groupconsisting of arginine, glycine alanine, valine, leucine, isoleucine,proline, serine, threonine, phenylalanine, tyrosine, tryptophan,aspartate, glutamate, asparagine, glutamine, cysteine, and methionine;wherein the amino acid at position 27 is independently selected from thegroup consisting of lysine, glycine, alanine, valine, leucine,isoleucine, proline, serine, threonine, phenylalanine, tyrosine,tryptophan, aspartate, glutamate, asparagine, glutamine, cysteine, andmethionine; wherein the amino acid at position 30 is independentlyselected from the group consisting of tyrosine, serine, lysine,arginine, histidine, aspartate, glutamate, asparagine, glutamine, andcysteine; wherein the amino acid at position 50 is independentlyselected from the group consisting of glutamic acid, lysine, arginine,histidine, glycine, and alanine; wherein the amino acid at position 59is independently selected from the group consisting of tryptophan,serine, lysine, arginine, histidine, aspartate, glutamate, asparagine,glutamine, and cysteine; and wherein the amino acid at position 60 isindependently selected from the group consisting of valine, serine,lysine, arginine, histidine, aspartate, glutamate, asparagine,glutamine, and cysteine. Such MDC polypeptide analogs are specificallycontemplated to modulate the binding characteristics of MDC to chemokinereceptors and/or other molecules (e.g., heparin, glycosaminoglycans,erythrocyte chemokine receptors) that are considered to be important inpresenting MDC to its receptor. In one preferred embodiment, MDCpolypeptide analogs of the invention comprise amino acids 1 to 69 of SEQID NO: 25.

The following additional analogs have been synthesized and also areintended as aspects of the invention: (a) a polypeptide comprising asequence of amino acids identified by positions 1 to 70 of SEQ ID NO:30; (b) a polypeptide comprising a sequence of amino acids identified bypositions 9 to 69 of SEQ ID NO: 2; (c) a polypeptide comprising asequence of amino acids identified by positions 1 to 69 of SEQ ID NO:31; and (d) a polypeptide comprising a sequence of amino acidsidentified by positions 1 to 69 of SEQ ID NO: 32.

As set forth in detail below, experimental data indicates that theaddition of as few as one additional amino acid at the amino terminus ofhuman mature MDC is sufficient to confer useful MDC inhibitoryproperties to the resultant analog. Thus, all amino terminal additionanalogs are contemplated as an aspect of the invention. Such additionanalogs include the addition of one or a few randomly selected aminoacids; the addition of common tag sequences (e.g., polyhistidinesequences, hemagglutinin sequences, or other sequences commonly used tofacilitate purification); and chemical additions to the amino terminus(e.g., the addition of an amino terminal aminooxypentane moiety). SeeProudfoot et al., J. Biol. Chem., 271:2599-2603 (1996); Simmons et al.,Science, 276 (5310): 276-279 (1997).

Also as set forth in detail below, evidence exists that mature MDC iscleaved in vivo by a dipeptidyl amino peptidase, resulting in anMDC(3-69) form that exhibits at least some activities antagonistic toMDC. An additional aspect of the invention includes analogs wherein theproline at position 2 of a mature MDC (e.g., human, murine, and rat MDC)is deleted or changed to an amino acid other than proline. Such analogsare collectively referred to as “MDCΔPro₂ polypeptides.” Those MDCΔPro₂polypeptides that retain MDC biological activities are contemplated asuseful in all indications wherein mature MDC is useful; and are expectedto be less susceptible to activity-destroying depeptidyl aminopeptidases that recognize and cleave the sequence NH2-Xaa-Pro- (e.g.,CD26). Those MDCΔPro₂ polypeptides that lack MDC biological activitiesare contemplated as being used as MDC inhibitors.

It will be appreciated that, while the foregoing analogs were oftendescribed with reference to human mature MDC, similar analogs of othervertebrate MDC's, especially mammalian MDC's, also are contemplated asaspects of the invention.

It also will be appreciated that it may be advantageous to express MDCor MD analogs as fusions with immunoglobulin-sequences, human serumalbumin sequences, or other sequences, or to perform other standardchemical modifications, for the purpose of extending the serum half-lifeof the MDC or MDC analog. See, e.g., Yeh et al., Proc. Nat'l. Acad Sc.U.S.A 89(5): 1904-1908 (1992); Sambrook et al, supra. The definition ofpolypeptides of the invention is intended to encompass suchmodifications.

In related aspects, the invention provides purified and isolatedpolynucleotide encoding such MDC polypeptide analogs, whichpolynucleotides are useful for, e.g. recombinantly producing the MDCpolypeptide analogs; plasmid and viral vectors incorporating suchpolynucleotides, and prokaryotic and eukaryotic host cells stablytransformed with such DNAs or vectors.

In another aspect, the invention includes antibody substances (e.g.,monoclonal a

polyclonal antibodies, single chain antibodies, chimeric or humanizedantibodies, antigen-bind

fragments of antibodies, and the like) which are immunoreactive with MDCpolypeptides a

polypeptide analogs of the invention. Such antibodies are useful, e.g.,for purifying polypeptides of the present invention, for quantitativemeasurement of endogenous MDC in a host, e.g., using well-known ELISAtechniques, and for modulating binding of MDC to its receptor(s). Tinvention further includes hybridoma cell lines that produce antibodysubstances of the invention Exemplary antibodies of the inventioninclude monoclonal antibodies 252Y and 252Z, which

produced by hybridoma cell line 252Y and hybridoma cell line 252Z,respectively. The hybrido

cell lines are themselves aspects of the invention, and have beendeposited with the American Type Culture Collection (ATCC Accession Nos.HB-12433 and HB-12434, respectively Another exemplary antibody of theinvention is monoclonal antibody 272D, which is produced by hybridomacell line 272D (itself an aspect of the invention and deposited with theAmerican Type Culture Collection (ATCC Accession No. HB-12498).

Recombinant MDC polypeptides and polypeptide analogs of the inventionmay utilized in a like manner to antibodies in binding reactions, toidentify cells expressing receptor of MDC and in standard expressioncloning techniques to isolate polynucleotides encoding receptor(s). SuchMDC polypeptides, MDC polypeptide analogs, and MDC receptor polypeptidesare useful for modulation of MDC chemokine activity, and foridentification of polypeptide and chemical (e.g., small molecule) MDCagonists and antagonists.

Additional aspects of the invention relate to pharmaceutical utilitiesof MDC polypeptides and polypeptide analogs of the invention. Forexample, MDC has been shown to modulate leukocyte chemotaxis. Inparticular, MDC has been shown to induce macrophage chemotaxis and toinhibit monocyte chemotaxis. Thus, in one aspect, the invention includesa method for modulating (e.g., up-regulating or down-regulating)leukocyte chemotaxis in a mammalian host comprising the step ofadministering to the mammalian host an MDC polypeptide or polypeptideanalog of the invention, wherein the MDC polypeptide or MDC polypeptideanalog modulates leukocyte chemotaxis in the host. In preferred methods,the leukocytes are monocytes and/or macrophages. For example,empirically determined quantities of MDC are administered (e.g., in apharmaceutically acceptable carrier) to induce macrophage chemotaxis orto inhibit monocyte chemotaxis, whereas inhibitory MDC polypeptideanalogs are employed to achieve the opposite effect.

In another aspect, the invention provides a method for palliating aninflammatory or other pathological condition in a patient, the conditioncharacterized by at least one of (i) monocyte chemotaxis toward a siteof inflammation in said patient or (ii) fibroblast cell proliferation,the method comprising the step of administering to the patient atherapeutically effective amount of MDC. In one embodiment, atherapeutically effective amount of MDC is an amount capable ofinhibiting monocyte chemotaxis. In another embodiment, a therapeuticallyeffective amount of MDC is an amount capable of inhibiting fibroblastcell proliferation. Such therapeutically effective amounts areempirically determined using art-recognized dose-response assays.

As an additional aspect, the invention provides a pharmaceuticalcomposition comprising an MDC polypeptide or polypeptide analog of theinvention in a pharmaceutically acceptable carrier. Similarly, theinvention relates to the use of a composition according to the inventionfor the treatment of disease states, e.g., inflammatory disease states.In one embodiment, the inflammatory disease state is characterized bymonocyte chemotaxis toward a site of inflammation in a patient havingthe disease state. In another embodiment, the disease state ischaracterized by fibroblast cell proliferation in a patient having thedisease state.

MDC induced chemotaxis of natural killer cells (NK) can lead to enhancedcytotoxicity of targeted NK cells against carious forms of cancers.These forms of cancers include all solid tumor and cancerous cells foundin various organs and skin (e.g., breast, ovarian, prostate, kidney,lung, pancreas, liver and bone cancers). NK cells also play an importantrole in antibody-dependent cell-mediated cytotoxicity. Stimulation ofthis process with MDC or MDC agonists would lead to improved immuneresponse to tumors. [See generally Immunology (Ed. Kuby, J.) pp 304-6,W.H. Freeman and Co., New York, N.Y. (1992)]. Similarly, NK cells leadto viral immunity. MDC may be used to potentiate resistance to commonviral diseases (e.g. influenza and rhinoviruses) by stimulating NKconferred viral immunity by stimulating antigen-specific T_(H) memorycells. [Immunology Ed. Kuby J. pp 420425, W.H. Freeman and Co. New York,N.Y. (1992)]. “Treatment” as used herein includes both prophylactic andtherapeutic treatment.

The apparent optimal concentration of mature MDC in receptor binding andchemotaxis experiments is about 10 ng/ml. Thus, for therapeutic methodsinvolving the systemic administration of MDC (or MDC analogs retaining adesired MDC biological activity), doses and dosing schedules arepreferably selected to maintain circulating concentrations in blood ofabout 0.1-10 ng/ml. Preferred approaches for preparing a dose andmaintaining such levels in the bloods include administration of MDC in abolus fashion, so as to administer approximately 0.1-10 mg of MDC. Thisadministration is repeated in order to maintain the stated bloodconcentration. For example, MDC is stable at 1 mg/ml inphosphate-buffered saline (PBS) and is administered to experimentalanimals using this formulation. This formulation, either liquid orlyophilized and reconstituted, is suitable for human parenteral use,e.g., via intravenous injection. Other formulations can be devised toconcentrate the protein drug and stabilize it for use years after itspreparation. [See, e.g., Stability and Characterization of Protein andPeptide Drugs; Case Histories, Wang Y J and Pearlman R. (Eds.), PlenumPress, New York (1993) (describing methods for the preparation ofcytokines and other similar protein drug formulations by the inclusionof a variety of excipients to maintain solubility and stability andminimize aggregation)]. Exemplary excipients include citrate, EDTA,detergents of the Tween family, zwittergent family, or pluronic family,and amino acids such as cysteine to maintain the proper oxido-reductantstate.

In a second preferred approach, MDC is administered using any of anumber of drug delivery methods that are known in the art to facilitateslow-release of the bioactive product. This can be accomplished aseasily as employing intramusculature administration [see for example M.Groves in Parental Technology Manual, Second edition, M.J. Groves (Ed.),Interpharm Press, Inc., Prairie View, Ill., pp. 6-7 (1988)] to cause theMDC to be adsorbed into the blood stream over a delayed period of time.Alternatively, the MDC product can be delivered using a number of drugdelivery methods [see for a general review LM Sanders, in Peptide andProtein Drug Delivery, V. H. L. Lee (Ed.), Marcel Dekker, Inc., NewYork, pp. 785-806 (1991)]. For example, MDC is incorporated intobiodegradable microspheres, such as poly(lactic-co-glycolic acid ofPLGA) microspheres as shown using Human Growth Hormone, [Tracy,Biotechnol. Progress, 14: 108-115 (1988)], or leuprolide acetatemicrospheres [Okada et al., Pharm. Res, 8: 787-791 (1991)] which canpermit administrations as infrequently as once monthly. A variety ofother drug delivery approaches will be apparent to those in the art,including dry powder formulations suitable for inhalation made availableby Inhale Corporation, Palo Alto, Calif., and transdermal delivery madeavailable by Alza Corporation, Palo Alto, Calif.

It will also be apparent from the teachings herein relating to thevarious activities of MDC that modulators of MDC activities, to inhibitthe effects of endogenously-produced MDC and/or to promote theactivities of endogenously-produced or exogenously administered MDC,have therapeutic utility. Such modulators typically include smallmolecules, peptides, chemokine fragments and analogs, and/or antibodysubstances. MDC inhibitors interfere with MDC signal transduction, e.g.,by binding MDC molecules, by competitively or non-competitively bindingMDC receptors on target cells, and/or by interfering with signaltransduction in the target cells downstream from the chemokinereceptors. Thus, in another aspect, the invention provides assays toscreen putative chemokine modulators for modulating activity. Modulatorsidentified by methods of the invention also are considered aspects ofthe invention:

In one embodiment, the invention provides a method for identifying achemical compound having MDC modulating activity comprising the stepsof: (a) providing first and second receptor compositions comprising MDCreceptors; (b) providing a control composition comprisingdetectably-labeled MDC; (c) providing a test composition comprisingdetectably-labeled MDC and further comprising the chemical compound; (d)contacting the first receptor composition with the control compositionunder conditions wherein MDC is capable of binding to MDC receptors; (e)contacting the second receptor composition with the test compositionunder conditions wherein MDC is capable of binding to MDC receptors; (f)washing the first and second receptor compositions to removedetectably-labeled MDC that is unbound to MDC receptors; (g) measuringdetectably-labeled MDC in the first and second receptor compositions;and (h) identifying a chemical compound having MDC modulating activity,wherein MDC modulating activity is correlated with a difference indetectably-labeled MDC between the first second receptor compositions.

As reported herein, the chemokine receptor CCR4 has been demonstrated tobe a high affinity receptor for MDC. Thus, in a preferred embodiment ofthe foregoing method, the first and second receptor compositionscomprise the MDC receptor that is CCR4. Since CCR4 is a membraneprotein, a preferred embodiment for practicing the method is one whereinthe first and second receptor compositions comprise CCR4-containing cellmembranes derived from cell that express CCR4 on their surface. The cellmembranes may be on intact cells, or may constitute an isolated fractionof cells that express CCR4. Cells that naturally express CCR4 and cellsthat have been transformed or transfected to express CCR4 recombinantlyare contemplated. In an alternative embodiment, cells (e.g.,eosinophils) that express an MDC receptor other than CCR4 are used toprovide the composition comprising MDC receptors.

In a related aspect, the invention provides a method for identifying amodulator of binding between MDC and CCR4, comprising the steps of: (a)contacting MDC and CCR4 both in the presence of, and in the absence of,a putative modulator compound; (b) detecting binding between MDC andCCR4; and (c) identifying a putative modulator compound in view ofdecreased or increased binding between MDC and CCR4 in the presence ofthe putative modulator, as compared to binding in the absence of theputative modulator. The contacting is performed, for example, bycombining MDC with cell membranes that contain CCR4, in a bufferedaqueous suspension.

In one embodiment, the method is performed with labeled MDC. In step(b), binding between MDC and CCR4 is detected by detecting labeled MDCbound to CCR4. In a preferred embodiment, the contacting step comprisescontacting a suspension of cell membranes comprising CCR4 with asolution containing MDC. In a highly preferred embodiment, the methodfurther comprises the steps of recovering the cell membranes from thesuspension after the contacting step (e.g., via filtration of thesuspension); and washing the cell membranes prior to the detecting stepto remove unbound MDC.

In an alternative embodiment, the method is performed with a host cellexpressing CCR4 on its surface. In step (b), binding between MDC andCCR4 is detected by measuring the conversion of GTP to GDP in the hostcell.

In yet another alternative embodiment, the method is performed with ahost cell that expresses CCR4 on its surface, and binding between MDCand CCR4 expressed in the host cell is detected by measuring cAMP levelsin the host cell.

It will be appreciated that assays for modulators such as thosedescribed above are often performed by immobilizing (e.g., on a solidsupport) one of the binding partners (e.g., MDC or a fragment thereofthat is capable of binding CCR4, or CCR4 or a fragment thereof that iscapable of binding MDC). In a preferred variation, the non-immobilizedbinding partner is labeled with a detectable agent. The immobilizedbinding partner is contacted with the labeled binding partner in thepresence and in the absence of a putative modulator compound capable ofspecifically reacting with MDC or CCR4; binding between the immobilizedbinding partner and the labeled binding partner is detected; andmodulating compounds are identified as those compounds that affectbinding between the immobilized binding partner and the labeled bindingpartner.

In yet another embodiment, the invention provides a method foridentifying a chemical compound having MDC modulating activity,comprising the steps of: (a) providing first and second receptorcompositions comprising MDC receptors; (b) contacting the first receptorcomposition with a control composition comprising detectably-labeledMDC; (c) contacting the second receptor composition with a testcomposition comprising detectably-labeled MDC and further comprising thechemical compound; (d) washing the first and second receptorcompositions to remove detectably-labeled MDC that is unbound to MDCreceptors; (e) measuring detectably-labeled MDC in the first and secondreceptor compositions after the washing; and (f) identifying a chemicalcompound having MDC modulating activity, wherein MDC modulating activityis correlated with a difference in detectably-labeled MDC between thefirst and the second receptor compositions.

In yet another embodiment, MDC binding to its receptor is measured bymeasurement of the activation of a reporter gene that has been coupledto the receptor using procedures that have been reported in the art forother receptors. See, e.g., Himmler et al., Journal of ReceptorResearch, 13:79-94 (1993).

MDC-binding fragments of high affinity receptors of MDC are specificallycontemplated as inhibitor compounds of the invention; antibodies to suchreceptors also are contemplated as inhibitor compounds of the invention.

As taught herein in detail, MDC stimulates eosinophil chemotaxis througha pathway that apparently does not involve the chemokine receptor CCR4.This discovery provides for the design of assays to identify modulatorsof MDC activity that have specificity for CCR4-mediated activitieswithout affecting MDC-induced stimulation of eosinophils, and viceversa.

For example, in one embodiment, the invention provides a method foridentifying a modulator of binding between MDC and eosinophils,comprising the steps of: (a) contacting MDC and a composition comprisingan MDC receptor that is expressed on eosinophil cell membranes in thepresence and in the absence of a putative modulator compound; (b)detecting binding between MDC and the composition; and (c) identifying aputative modulator compound in view of decreased or increased bindingbetween MDC and the composition in the presence of the putativemodulator, as compared to binding in the absence of the putativemodulator.

To identify modulators with eosinophil-specificity, the method, in apreferred embodiment, further comprising the steps of: (d) contactingMDC and a composition comprising CCR4 in the presence and in the absenceof the putative modulator compound; (e) detecting binding between MDCand CCR4; (f) identifying a putative modulator compound in viewdecreased or increased binding between MDC and CCR4 in the presence ofthe putative modulator, as compared to binding in the absence of theputative modulator; and (g) selecting modulator identified in step (c)as causing increased or decreased binding and identified in step (f) asfailing to cause increased or decreased binding. To identify modulatorswith specificity towards CCR4, in step (g) one selects a modulatoridentified in step (f) as causing increased or decreased binding andidentified in step (c) as failing to cause increased or decreasedbinding.

MDC's involvement in various aspects of immune responses is described indetail below. Based on the involvement of MDC in immune response, theadministration of MDC antagonists is indicated, for example, in thetreatment anaphylaxis [Brown, A. F., J. Accid Emerg. Med., 12(2):89-100(1995)], shock [Brown (1995) supra], ischemia, reperfusion injury andcentral ischemia [Lindsberg et al., Ann. Neurol, 30(2):117-129 (1991)],atherogenesis [Handley et al., Drug Dev. Res., 7:361-375 (1986)],Crohn's disease [Denizot et al., Digestive Diseases and Sciences,37(3):432-437 (1992)], ischemic bowel necrosis/necrotizing enterocolitis[Denizot et al. (1992), supra, and Caplan et al., Acta Pediat. Suppl.,396:11-17 (1994)], ulcerative colitis (Denziot et al. (1992), supra),ischemic stroke [Satoh et al., Stroke, 23:1090-1092 (1992)], ischemicbrain injury [Lindsberg et al., Stroke, 21:1452-1457 (1990) andLindsberg et al. (1991), supra], systemic lupus erythematosus [Matsuzakiet al., Clinica Chimica Acta, 210:139-144 (1992)], acute pancreatitis[Kald et al., Pancreas, 8(4):440-442 (1993)], septicemia (Kald et al.(1993), supra), acute post-streptococcal glomerulonephritis [Mezzano etal., J. Am. Soc. Nephrol., 4:235-242 (1993)], pulmonary edema resultingfrom IL-2 therapy [Rabinovichi et al., J. Clin. Invest., 89:1669-1673(1992)], ischemic renal failure [Grino et al, Annals of InternalMedicine, 121(5):345-347 (1994)]; pre-term labor [Hoffman et al., Am. J.Obstet. Gynecol., 162(2):525-528 (1990) and Maki et al., Proc. Natl.Acad. Sci. USA, 85:728-732 (1988)], adult respiratory distress syndrome[Rabinovichi et al., J. Appl. Phsiol., 74(4):1791-1802 (1993); Matsumotoet al., Clin. Exp. Phannocol. Physiol., 19:509-515 (1992); andRodriguez-Roisin et al., J. Clin Invest., 93:188-194 (1994)].“Treatment” as used herein includes both prophylactic and therapeutictreatment.

MDC acts as a chemoattractant for T_(H)2 differentiated memory cells,which produce the cytokines IL-4, IL-5, IL-10 and others. It is expectedthat, in some instances, MDC leads to an immune state in which T_(H)1cytokine driven responses are reduced. In such instances, antagonism ofMDC would lead to a state in which T_(H)1 cytokine driven responses areenhanced. Modulation of the T_(H)1-T_(H)2 balance may lead to enhanced“immune surveillance,” and improved eradication of viral and parasiticinfections. Administration of MDC antagonists of the invention tomammalian subjects, especially humans, for the purposes of amelioratingpathological conditions associated with undesirable or excessive T_(H)2responses and/or less-than-desirable T_(H)1 responses are contemplatedas additional aspects of the invention. Administration of sufficient MDCantagonists to substantially reduce endogenous IL-10, a T_(H)1 immunesuppressing cytokine, would lead to enhanced cytotoxic T-lymphocytemediated immunity and immune surveillance [see Muller et al., J. Infect.Dis., 177: 586-94 (1998); Kenney et al., J. Infect. Dis., 177: 815-9(1998)]. In these situations an effective dose and dosing schedule canbe determined by monitoring circulating IL-10 levels and increasing thedose and frequency of administration to reduce IL-10 levels to nearnormal levels. Treatment of chronic or persistent viral infections andparasitic infections is specifically contemplated, especially incombination with other antiviral or anti-parasitic infectiontherapeutics. Similarly, treatment or prevention of graft failure orgraft rejection with MDC antagonists is contemplated. The administrationof MDC antagonists is indicated, for example, in Leishmaniasis [Li etal., Infect. Immunol., 64:5248-5254 (1996); Krishnan et al., J.Immunol., 156(2):653-62 (1996)], opportunistic lung infections in cysticfibrosis patients [Moser et al., APMIS, 105(11):83842 (1997)], to delayHIV-1 induced immunodeficiency [Berger et al., Res. Virol.,147(2-3):103-108 (1996); Barker et al., Proc. Natl. Acad. Sci. USA,92(24):11135-9 (1995); Jason et al., J. Acquir. Immune. Defic. SyndromeRetrovirol., 10(4): 471-6 (1995); Maggi et al., J. Biol. Regul. Homeost.Agents, 9(3): 78-81 (1995)], chronic interstitial lung disease [Kunkelet al., Sarcoidosis Vasc. Diffuse Lung Dis., 13: 120-128 (1996)], inneurological disorders associated with a T_(H)2 response [Windhagen etal., Chem. Immunol., 63: 171-86 (1996); Bai et al., Clin. Immunol.Immunopathol., 83(2): 117-2 (1997)], colorectal cancer [Pellegrini etal., Cancer Immunol. Immunother., 42(1): 1-8 (1996) viral infection, forexample various species of herpes and hepatitis [Spruance et al.,Antiviral Res., 28(1): 39-55 (1995); Pope et al, J. Immunol., 156(9):3342-9 (1996); Bartoletti et al., Gastroenterol., 112(1): 193-199(1997)], candidiasis and other fungal infections [Spaccapelo et al., J.Immunol., 155(3): 1349-60 (1995); Fidel et al, J. Infect. Dis., 176(3):728-39 (1995 ) Cenci et al, J. Infect Dis., 171(5): 1279-88 (1995)],chronic pneumonia [Johansen et al, Behring Inst. Mitt., 98: 269-73(1997)], solid tumor cancer [Khar et al., Cytokines Mol Ther., 2(1):39-46 (1996)], Bordella pertussis respiratory infection [Ryan et al, JInfect Dis., 175(5): 1246-50 (1997)], systemic lupus erythrematosus[Segal et al., J. Immunol., 158(6): 2648-53 (1997)], Bullous pemphigoiddpathogenesis (Deptia et al., Arch Dermatol Res., 289(12): 667-70(1997)], glomerulonephritis [Kitching et al, Kidney Int., 53(1): 112-8(1998); Huang et al, J. Am Soc. Neprol., 8(7): 1101-8 (1997); Tipping etal., Eur. J. Immunol., 27(2): 515-21 (1997)], pulmonary respiratorysyncytial virus infection [Hussell et al., Eur. J. Immunol.; 27(12):3341-9 (1997 )], complications of trauma associated with surgical stress[Decker et al., Surgery, 119(3): 316-25 -(1996)], celiac disease [Karbanet al., Isr. J. Med Sci., 33(3): 209-14 (1997)], Gulf War syndrome [Rooket al., Lancet, 349(9068): 1831-3 (1997)], ameobocyte infection, forexample Plasmodium falciparum [Elghazali et al., Clin. Exp. Immunol.,109(1): 84-9 (1997)] and schistosoma mansoni [Wolowczuk et al.,Immunol., 91(1): 35-44 (1997)], and B-cell lymphoma especiallymucosa-associated lymphoid tissue type [Greiner et al., Am J. Pathol.,150(5): 1583-93 (1997)]. “Treatment” as used herein includes bothprophylactic and therapeutic treatment.

In fact, the expression pattern of MDC (or TARC) and its receptor CCR4provide a unique indication for MDC in vivo in inducing a cellularcomplex (e.g., dendritic and/or macrophage cells, T_(H)2antigen-specific memory cells, and antigen-specific B cells) geared toproducing a strong humoral immune response. The induced complex iscontemplated to produce antigen-specific antibodies and T_(H)2-specificcytokines (IL-2, IL-4, IL-5, and/or IL-10) and additional chemokines,including additional MDC, with local concentrations of the chemokinesand cytokines that potentiate the activity of the complex possibly beingquite high. The cellular complex is specifically contemplated to beinvolved in the establishment of a humoral response to “recallantigens,” since another chemokine/receptor pair (MP3α/CCR6) appears tobe specific for “naive” responses to new antigens. Thus, administrationof SMC or MDC agonists for the purpose of inducing or augmenting aresponse to “recall antigens” is specifically contemplated as an aspectof the intention. Similarly, administration of MDC antagonists isindicated when suppression of such an immune response is desired.Administration of MDC antagonists to treat conditions and disordersmediated (directly or indirectly) by T_(H)2 cell migration, includingbut not limited to autoimmune conditions, lupus erythematosus, multiplesclerosis, scleroderma, asthma, and atopic allergy, is specificallycontemplated.

With respect to any of the conditions, disorders, and disease statesidentified in the preceding paragraphs, an exemplary method of treatmentcomprises the steps of identifying a human subject in need oftherapeutic or prophylactic treatment for one of the above-identifiedconditions, disorders, or disease states; and administering to the humansubject a therapeutically or prophylactically effective amount of an MDCantagonist compound. By “therapeutically effective amount” is meant adose and dosing schedule that is sufficient to cure the disease state,or to reduce the symptoms or severity of the disease state. By“prophylactically effective amount” is meant a dose and dosing schedulethat is sufficient to reduce the likelihood of occurrence of a diseasestate, or delay its onset, relative to human subjects that areconsidered to have equivalent risk of developing the disease state butwhom are not treated with an MDC antagonist. Therapeutically effectiveamounts are readily determined by dose-response studies that areconventionally performed in the art.

In one highly preferred embodiment, the invention includes a method ofinhibiting proliferation of a mammalian immunodeficiency viruscomprising the step of contacting mammalian cells that are infected witha mammalian immunodeficiency virus with a composition comprising anMDC-IV antagonist compound or TARC-IV antagonist compound, in an amounteffective to inhibit proliferation of said virus in said cells. Thefamily of mammalian immunodeficiency viruses is intended to includehuman immunodeficiency viruses, such as strains of HIV-1 and HIV-2, andanalogous viruses known to infect other mammalian species, including butnot limited to simian and feline immunodeficiency viruses. The methodcan be performed in vitro (e.g., in cell culture), but preferably isperformed in vivo by administering the antagonist to an infectedsubject, e.g., an HIV-infected human subject. (In yet anotherembodiment, the method is performed prophylacticly on a subject at riskof developing an HIV infection, e.g., due to the subject's likelihood ofexposure to contaminated blood samples, contaminated needles, orintimate exposure to an HIV-infected person.)

The term “MDC-IV antagonist compound” refers to compounds thatantagonize the apparent Immunodeficiency Virus-proliferative effects ofMDC in infected cells. Thus, the term “MDC-IV antagonist compound” ismeant to include any compound that is capable of inhibitingproliferation of the immunodeficiency virus in a manner analogous toeither the inhibition reported herein for MDC neutralizing antibodies orthe inhibition reported herein for certain MDC analogs (e.g., analogshaving amino terminal additions or truncations). For example, anti-MDCantibodies are highly preferred MDC-IV antagonist compounds. Fortreatment of humans infected with an HIV virus, humanized antibodies arehighly preferred. Similarly, polypeptides that comprise anantigen-binding fragment of an anti-MDC antibody and that are capable ofbinding to MDC are preferred MDC-IV antagonist compounds.

As described elsewhere herein in greater detail, amino-terminaltruncations of mature human MDC(1-9) possess antiproliferative activityagainst HIV-1. Thus, another set of preferred MDC-IV antagonistcompounds are polypeptides whose amino acid sequence consists of aportion of the amino acid sequence set forth in SEQ ID NO: 2 sufficientto bind to the chemokine receptor CCR4, said portion having anamino-terminus between residues 3 and 12 of SEQ ID NO: 2 (i.e., analogslacking at least three amino acids from the amino terminus of MDC(1-69).Amino terminal deletion analogs that have been further modified, e.g.,by including an oligopeptide tag to facilitate purification, or byincluding an initiator methionine for bacterial expression, are alsocontemplated.

Amino-terminal additions to mature MDC also result in analogs possessingantiproliferative activity against HIV-1. Thus, another set of preferredMDC-IV antagonist compounds are polypeptides that comprise a mature MDCsequence (e.g., amino acids 1-69 of SEQ ID NO: 1), and that furthercomprise a chemical addition to the amino terminus of the mature MDCsequence to render said polypeptide antagonistic to MDC. Additions ofadditional amino acids and other chemical moieties are contemplated.

It will further be appreciated that substitution of amino acids in amature MDC sequence (especially substitutions in the amino terminus ofmature MDC) may be expected, in some instances, to result in analogspossessing antiproliferative activity against HIV-1. Such analogs alsoare intended MDC-IV antagonist compounds, and are identifiable using HIVproliferation assays described herein.

It is postulated that MDC's HIV-proliferative effects are mediated, atleast in part, through the chemokine receptor CCR4. Thus, the family ofMDC-IV antagonist compounds includes polypeptides that comprise the C-Cchemokine receptor 4 (CCR4) amino acid sequence set forth in SEQ ID NO:34 or that comprise a continuous fragment thereof that is capable ofbinding to MDC or TARC. Such polypeptides are expected to bindendogenous MDC and thereby inhibit HIV proliferation in a manneranalogous to anti-MDC antibodies. Also contemplated are anti-CCR4antibodies, which are expected to block MDC-CCR4 interactions, therebyinhibiting MDC-induced HIV proliferation.

As described herein in detail, the chemokine TARC possesses sequencesimilarity to MDC, possesses various overlapping biological activities,and, like MDC, binds to the chemokine receptor CCR4. These similaritiessuggest that compounds that inhibit TARC-CCR4 interactions will also beuseful for inhibiting proliferation of immunodeficiency viruses.Compounds that inhibit TARC-induced proliferation of such viruses arecollectively referred to as “TARC-IV antagonist compounds.” Suchcompounds include anti-CCR4 antibodies, anti-TARC antibodies (especiallyhumanized versions); and polypeptides that are capable of binding toTARC and that comprise an antigen-binding fragment of an anti-TARCantibody.

It is also contemplated that modifications to the amino terminus ofmature TARC polypeptides will result in TARC-IV antagonist compounds, ina manner analogous to what has been reported herein for MDC analogs.Thus, TARC-IV antagonist compounds for use in methods of the inventioninclude polypeptides that have an amino acid sequence consisting of aportion of the amino acid sequence set forth in SEQ ID NO: 43 that issufficient to bind to the chemokine receptor CCR4, said portion havingan amino-terminus between residues 1 and 10 of SEQ ID NO: 43.Polypeptide comprising mature TARC sequences, and further comprisingchemical additions to the amino terminus to render the polypeptideantagonistic to TARC also are contemplated. Polypeptides comprising themature TARC amino acid sequence, into which substitutions have beenintroduced to confer HIV antiproliferative activity, also arecontemplated as TARC-IV antagonist compounds.

In another highly preferred embodiment, the invention includes a methodof inhibiting platelet aggregation in a mammalian subject (especially ahuman subject) comprising the step of administering to a mammaliansubject a composition comprising an MDC-PA antagonist compound orTARC-PA antagonist compound, in an amount effective to inhibit plateletaggregation in the subject. Such methods may be performed fortherapeutic purposes, e.g., in patients suffering from undesirable bloodclotting, or for prophylactic purposes on a subject at risk ofdeveloping undesirable blood clotting or coagulation. Such patientswould include, e.g., patients who have previously suffered myocardialinfarction or stroke or other clotting disorders, or who are deemed tobe at high risk for developing such conditions.

The term “MDC-PA antagonist compound” refers to compounds thatantagonize the apparent Platelet Aggregating effects of MDC. Thus, theterm “MDC-PA antagonist compound” is meant to include any compound thatis capable of inhibiting platelet aggregation that is observable afteradministration of MDC to a mammalian subject (e.g., to a mouse or rat).Those compounds described above as MDC-IV antagonist compounds arespecifically contemplated as MDC-PA antagonist compounds as well. Forexample, anti-MDC antibodies are highly preferred MDC-PA antagonistcompounds. For treatment of humans, humanized antibodies are highlypreferred. Similarly, polypeptides that comprise an antigen-bindingfragment of an anti-MDC antibody and that are capable of binding to MDCare preferred MDC-PA antagonist compounds. All MDC analogs that inhibitthe platelet aggregating effects of MDC also are preferred. Analogshaving additions, deletions, and/or substitutions in the amino terminusare specifically contemplated.

The structural and functional similarities between MDC and TARC reportedherein indicate that compounds that inhibit TARC-CCR4 interactions willbe useful for inhibiting platelet aggregation. Compounds that inhibitTARC-induced platelet aggregation are collectively referred to as“TARC-PA antagonist compounds.” Such compounds include anti-CCR4antibodies, anti-TARC antibodies (especially humanized versions);various TARC analogs described elsewhere herein, and polypeptides thatare capable of binding to TARC and that comprise an antigen-bindingfragment of an anti-TARC antibody.

As described herein in detail, the expression patterns of MDC and itsreceptor, CCR4, provide an indication for the use of MDC as an adjuvantin a vaccine. Thus, in another aspect, the invention includes a vaccinecomposition comprising an antigen of interest in a suitablepharmaceutical carrier, improved by the inclusion of MDC in the vaccinecomposition. The antigen of interest may be any composition intended togenerate a desirable immune response in a human or other animal. Suchcompositions would include, for example, killed or attenuated pathogensor antigenic portions thereof. In a related aspect, the inventionincludes a method of immunizing a human or animal, wherein theimprovement comprises administering MDC to the human or animal, eitherconcurrently or before or after administering an antigen of interest. Asexplained above, MDC is contemplated to preferentially augment an immuneresponse to “recall antigens.” Accordingly, in a preferred embodiment,MDC is included in a booster vaccine composition.

For any of the therapeutic indications and methods described above,another aspect of the invention relates to the use of indicatedcompounds (e.g., MDC, MDC fragments or analogs, MDC agonists, or MDCantagonists) for preparation of a medicament for the therapeuticindication. For example, the invention includes the use of an MDCantagonist for preparation of a medicament for suppressing a humoralresponse to recall antigens.

The foregoing aspects and numerous additional aspects will be apparentfrom the drawing and detailed description which follow.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a comparison of the amino acid sequence of human MDC (SEQ IDNO: 2) with the amino acid sequences of other, previously characterizedhuman C-C chemokines: MCP-3 [Van Damme et al., J. Exp. Med., 176:59(1992)] (SEQ ED NO: 18); MCP-1 [Matsushima et al., J. Exp. Med, 169:1485(1989)] (SEQ ID NO: 19); MCP-2 (mature form) [Van Damme et al., supra;Chang et al., Int. Immunol., 1:388 (1989)] (SEQ ID NO: 20 ); RANTES[Schall et al., J. Immunol., 141:1018 (1988)] (SEQ ID NO: 21); MIP-1β[Brown et al., J. Immunol., 142:679 (1989)] (SEQ ID NO: 22); MIP-1α[Nakao et al, Mol. Cell Biol., 10:3646 (1990)] (SEQ ID NO: 23); andI-309 [Miller et al., J. Immunol., 143:2907 (1989 )](SEQ ID NO: 24). Aslash “/” marks the site at which putative signal peptides are cleaved.Dashes are inserted to optimize alignment of the sequences.

FIG. 2 is a graph depicting the chemotactic effect (measured influorescence units) of increasing concentrations of MDC on humanmononuclear cell migration in a chemotaxis assay. Closed circles showthe response of human mononuclear cells derived from the cell lineTHP-1. The open diamond shows the response to the positive control,zymosan activated serum (ZAS).

FIG. 3 is a graph depicting the chemotactic effect (measured influorescence units) of increasing concentrations of MDC on humanpolymorphonuclear (pmn) leukocyte migration. Closed circles showresponse to MDC, and an open diamond shows the response to the positivecontrol, IL-8.

FIG. 4 is a graph depicting the chemotactic effect (measured influorescence units) of increasing concentrations of MDC on macrophageand monocyte migration. Closed circles show the response to MDC ofmacrophages derived from the cell line THP-1. Open circles show theresponse to MDC of monocytes derived from the cell line THP-1.

FIG. 5 is a graph depicting the chemotactic effect (measured influorescence units) of increasing concentrations of MDC on guinea pigperitoneal macrophage migration. Closed circles show the response ofmacrophages to MDC. An open triangle shows the response to the positivecontrol, zymosan activated serum (ZAS).

FIG. 6 is a graph depicting the chemotactic-inhibitory effect (measuredin fluorescence units) of increasing concentrations of MDC on THP-1monocyte migration induced by MCP-1. Closed circles depict thechemotactic-inhibitory effects of MDC where chemotaxis has been inducedby MCP-1. Open circles depict the chemotactic-inhibitory effects of MDCin a control experiment wherein only the basal medium (RPMI with 0.2%BSA (RBSA), no MCP-1 was employed. The zero point on the x axiscorresponds to the response of cells to MCP-1 and RBSA in the absence ofany MDC.

FIG. 7 is a graph depicting the effect (measured in counts per minute(cpm)) of increasing concentrations of MDC on fibroblast proliferation.Closed circles depict the proliferative response with purified MDC thatwas recombinantly produced in CHO cells (Example 10F). Open circlesdepict the response with chemically synthesized MDC (Example 11).

FIG. 8 schematically depicts the construction of mammalian expressionvector pDC1.

FIG. 9 depicts the nucleotide and deduced amino acid sequence (SEQ IDNOs: 39 and 40) of a S. cerevisiae alpha factor pre-pro/human MDC cDNAchimeric construct used to express human MDC in yeast.

FIG. 10 depicts the structure of plasmid pYGL/preproMDC, used to expresshuman MDC in yeast.

FIG. 11 depicts the inhibitory effects of the anti-MDC antibodies 252Yand 252Z on the binding of the fusion protein MDC-SEAP to the MDCreceptor designated CCR4. Binding (depicted as percent of maximalbinding) is plotted as a function of increased concentrations ofantibody

FIG. 12 depicts the inhibitory effects of the anti-MDC antibodies 252Yand 252Z on the MDC-induced chemotaxis of CCR4-transfected L1.2 cells.The number of cells observed migrating toward MDC in a standardchemotaxis assay are plotted as a function of increased concentrationsof antibody.

DETAILED DESCRIPTION

The present invention is illustrated by the following examples relatedto a human cDNA, designated MDC cDNA, encoding a novel C-C chemokinedesignated MDC (for “macrophage-derived chemokine”). More particularly,Example 1 describes the isolation of a partial MDC cDNA from a humanmacrophage cDNA library. Example 2 describes the isolation of additionalcDNAs from the cDNA library using the cDNA from Example 1 as a probe,one of these additional cDNAs containing the entire MDC coding sequence.Additionally, Example 2 presents a composite MDC cDNA nucleotidesequence and presents a characterization of the deduced amino acidsequence of the chemokine (MDC) encoded thereby. In Example 3,experiments are described which reveal the level of MDC gene expressionin various human tissues. The greatest MDC gene expression was observedin the thymus, with much weaker expression detectable in spleen and lungtissues. Example 4 describes more particularly the expression of the MDCgene during monocyte maturation into macrophages and during inducementof HL60 cell differentiation to a macrophage-like cell type.

Since MDC gene expression was detected in thymus and spleen in Example3, in situ hybridization studies were conducted to localize further theMDC gene expression in these tissues. Moreover, in situ hybridizationrevealed a correlation between elevated MDC gene expression in inflamedtissues, as exemplified using intestinal tissue from Crohn's diseasedpatients. These in situ hybridization experiments are described inExample 5.

Example 6 describes the recombinant production of MDC as a GST fusionprotein in prokaryotic cells, as well as the cleavage of the fusionprotein and purification of the recombinant MDC. Example 7 describesalternative DNA constructs useful for expression of recombinant MDCprotein, and describes the production of MDC by a bacterial hosttransformed with such a construct.

Example 8 provides experimental protocols for purification ofrecombinant MDC produced, e.g., as described in Example 7. Examples 9and 10 provide protocols for the recombinant production of MDC in yeastand mammalian cells, respectively. In addition, Example 10 providesadditional protocols for purification of recombinant MDC, and describesthe determination of the amino terminus of MDC recombinantly produced inmammalian cells. Example 11 describes production of MDC and MDCpolypeptide analogs by peptide synthesis. Certain preferred analogs arespecifically described in Example 11.

Examples 12-17 provide protocols for the determination of MDC biologicalactivities. For instance, Example 12 provides an assay of MDC effectsupon basophils, mast cells, and eosinophils. MDC-induced chemotaxis ofeosinophils is specifically demonstrated. Example 13 describes assays ofchemoattractant and cell-activation properties of MDC onmonocytes/macrophages, neutrophils, and granulocytes. MDC inducedmacrophage chemotaxix, but inhibited monocyte chemotaxis.

Examples 14-17 provide protocols for the determination of MDC biologicalactivities in vivo. Example 14 provides an MDC tumor growth-inhibitionassay. Examples 15 and 16 provide protocols for assaying MDC activityvia intraperitoneal and subcutaneous injection, respectively. Example 17provides protocols for determining the myelosuppressive activity of MDC.

Example 18 provides protocols for generating antibodies that arespecifically immunoreactive with MDC, including polyclonal, monoclonal,and humanized antibodies. Uses of the antibodies also are described.

Example 19 provides a calcium flux assay for determining the ability ofMDC to induce cellular activation.

Example 20 provides assays and experimental results relating to the HIVproliferative and anti-proliferative effects of human mature MDC and MDCantagonists.

Example 21 demonstrates the anti-proliferative effects of MDC onfibroblasts. Example 22 provides in vitro assays for the effects of MDCupon the proliferation of additional cell types. Example 23 provides anin vivo assay for determining the anti-proliferative effects of MDC onfibroblasts.

Example 24 describes the chromosomal localization of the human MDC gene.

Example 25 describes procedures which identified the CC chemokinereceptor “CCR4” as a high affinity binding partner of MDC. Examples 26and 27 provide assays for identifying MDC modulators.

Example 28 describes the isolation of cDNAs encoding rat, mouse, andmacaque MDC, and characterizes the MDC proteins encoded thereby. Example29 further characterizes selected MDC analogs.

Example 30 describes experiments that demonstrate that anti-MDCmonoclonal antibodies are effective for neutralizing biologicalactivities of MDC that were elucidated in other examples.

Example 31 describes experiments that demonstrate that MDC induceschemotaxis of T_(H)2 helper cells, a discovery with therapeuticimplications as discussed in Example 31 and elsewhere herein.

Example 32 describes platelet-aggregating activities of MDC, anddescribes the use of MDC and MDC antagonists to modulate plateletaggregation.

Example 33 provides exemplary assays to demonstrate the therapeuticefficacy of an MDC antagonist to modulate immune responses in amammalian host.

EXAMPLE 1 Isolation of a cDNA Encoding MDC

A partial cDNA for a new C-C chemokine, designated pMP390, was isolatedfrom a macrophage cDNA library as described in U.S. patent applicationSer. No. 08/939,107, filed Sep. 26, 1997, and in related internationalpublication number WO 96/40923, both of which are incorporated herein byreference. Sequence comparisons were performed on Dec. 14, 1994, by theBLAST Network Service of the National Center for BiotechnologyInformation (e-mail: “blast@ncbi.nlm.nih.gov”), using the alignmentalgorithm of Altschul et al., J. Mol. Biol., 215: 403-410 (1990). Thesequence analysis revealed that a portion of the isolated macrophagecDNA clone designated pMP390 contained a gene sequence havingapproximately 60-70% identity with previously-identified chemokinegenes, including the human MCP-3 gene and rat MIP-1β gene.

The 2.85 kb cDNA insert of pMP390 was subcloned into the vectorpBluescript SK⁻ (Stratagene, La Jolla Calif.) to facilitate completesequencing. The complete sequence of this pMP390 cDNA corresponds tonucleotides 73 to 2923 of SEQ ID NO: 1 (and to deduced amino acids −6 to69 of SEQ ID NO 2). The sequence that was originally compared todatabase sequences corresponds to nucleotides 73 to 610 of SEQ ID NO: 1.

EXAMPLE 2 Isolation of Additional cDNA Clones Having the Complete MDCCoding Sequence

Using the pMP390 cDNA clone isolated in Example 1, additional cDNAclones were isolated from the same human macrophage cDNA library, theseadditional cDNAs containing additional 5′ sequence and encoding thecomplete amino acid sequence of a macrophage derived chemokine. Theadditional cloning and sequencing is described in detail in U.S. Ser.No. 08/939,107 and WO 96/40923, incorporated herein by reference.

Of the additional clones, clones designated pNT390-12 and pMP390Bcontained the largest additional 5′ coding sequence, each extending anadditional 72 nucleotides upstream of the sequence previously obtainedfrom the cDNA clone pMP390. A composite DNA sequence, herein designatedMDC cDNA, was generated by alignment of the pMP390 and pMP390-12 cDNAsequences. This 2923 base pair composite cDNA sequence, and the deducedamino acid sequence of the chemokine MDC, are set forth in SEQ ID NOs: 1and 2, respectively.

Manual comparison of the deduced MDC amino acid sequence with sequencesof known chemokines indicates that the MDC cDNA sequence encodes a novelC-C chemokine ninety-three amino acids in length, sharing 28-34% aminoacid identity with other C-C chemokines (FIG. 1). As aligned in FIG. 1,MDC shares 29% amino acid identity with MCP-1 and MIP-1α, 28% identitywith MCP-2, 32% identity with I-309, 33% identity with MCP-3 and MIP-1β,and 34% identity with RANTES. Importantly, the four cysteine residuescharacteristic of the chemokines are conserved in MDC. Five additionalresidues also are completely conserved in the eight sequences presentedin FIG. 1.

The first 24 amino acids of the 93 amino acid MDC sequence arepredominantly hydrophobic and are consistent with von Heijne's rules[Nucleic Acids Res., 14: 4683-90 (1986)] governing signal cleavage.These features and the polypeptide comparison in FIG. 1 collectivelysuggest that the MDC cDNA encodes a twenty-four amino acid signalpeptide that is cleaved to produce a mature form of MDC beginning withthe glycine residue at position 1 of SEQ ID NO: 2. This prediction wasconfirmed by direct sequencing of MDC protein produced recombinantly inmammalian cells, as described below in Example 10. The MDC compositecDNA sequence shown in SEQ ID NO: 1 extends nineteen nucleotidesupstream of the predicted initiating methionine codon, and 2.6 kbdownstream of the termination codon.

EXAMPLE 3 Determination of MDC Gene Expression in Human Tissues

Northern blot analysis were conducted to determine the tissues in whichthe MDC gene is expressed.

A radiolabeled pMP390 5′ fragment which corresponds to the region of theMDC cDNA encoding the putative mature form of MDC plus 163 bases of theadjacent 3′ noncoding region was used to probe Multiple Tissue Northernblots (Clontech, Palo Alto, Calif.) containing RNA from various normalhuman tissues. The probe was denatured by boiling prior to use, and thehybridizations were conducted according to the manufacturer'sspecifications. Autoradiographs were exposed 5 days at −80° C. with 2intensifying screens.

The greatest MDC gene expression was observed in the thymus, with muchweaker expression detectable in spleen and lung tissues. Expression ofMDC in tissue from the small intestine was at even lower levels, and noexpression was detected in brain, colon, heart, kidney, liver, ovary,pancreas, placenta, prostate, skeletal muscle, testis, or peripheralblood leukocytes.

As discussed in detail below in Example 25, MDC is a ligand for the CCchemokine receptor CCR4, which receptor also has been reported to be aligand for the chemokine TARC. See Imai et al., J. Biol. Chem., 272:1503615042(1997). Like MDC, TARC is abundantly expressed in the thymus,with little expression observed in other tissues. More particularly,CCR4 is expressed on T cells, especially CD4⁺ T cells [See Imai et al.(1997), and Power el al., J. Biol. Chem., 270: 19495-19500 (1995)],while MDC and TARC are expressed by cells of the dendritic lineage whichform a major component of the thymic architecture. See Godiska et al.,J. Fxp. Med, 185: 1595-1604 (1997), incorporated herein by reference;and Imai et al., J. Biol. Chem., 271: 21514-21521 (1996). Theseexpression patterns suggest a biological activity of MDC, CCR4, and TARCin T cell development, since immature progenitor cells undergodifferentiation and expansion (leading to the establishment of the majorT cell lineages and the elimination of potentially autoreactive T cells)within the highly specialized microenvironment of the thymus. See vonBoehmer, Current Biology, 7: 308-310 (1997). The fact that MDC also isexpressed at high levels in cultured macrophages suggests an MDCactivity in the initiation and/or triggering of the immune response, byfacilitating the interaction of T cells with antigen-presenting cells atsites of inflammation.

These expression pattern data suggest therapeutic utilities of MDC (orMDC mimetics or agonists) to stimulate beneficial immune responses. Forexample, MDC, MDC agonists, or MDC mimetics may be administered toaugment/enhance T cell activation where T cell activation may bebeneficial. The use of MDC as an adjuvant in vaccine development or intumor immunotherapy is specifically contemplated.

Conversely, the expression pattern data also indicates a therapeuticutility for modulators of MDC's interaction with CCR4 in T cell-mediatedautoimmune diseases, including but not limited to psoriasis, graftversus host disease, and allograft rejection, and in T cell and/or Bcell mediated allergic responses.

EXAMPLE 4 MDC Gene Expression During Macrophage Maturation

Because the cDNAs encoding MDC were isolated from a human macrophagecDNA library, MDC gene expression during differentiation of monocytesinto macrophages was examined.

A

Human monocytes from a single donor were cultured on a series of tissueculture plates, and cells from one plate were harvested after 0, 2, 4 or6 days. See generally Elstad et al., J. Immunol. 140:1618-1624; Tjoelkeret al., Nature, 374:549-552 (1995). Under these conditions, themonocytes differentiated into macrophages by days 4-6 [Stafforini etal., J. Biol. Chem., 265: 9682-9687 (1990)].

A Northern blot of RNA (10 μg per lane) isolated from the cellsharvested at each time point was prepared and probed, using aradiaolabeled pMP390 fragment. No signal was detectable in RNA fromfreshly isolated monocytes, whereas a very strong signal was generatedfrom cells that had differentiated into macrophages after six days ofculture. Cells cultured for four days produced a much weaker signal,whereas the signal generated from cells cultured for two days could beseen only after prolonged exposure of the filter.

B

To confirm the expression of MDC in differentiated human macrophages,culture supernatants were analyzed by western blotting with anti-MDCmonoclonal antibodies produced as described below in Example 18. Severalplates of human macrophages were differentiated by growth on plastic foreight days in the presence of macrophage colony stimulating factor (0.5ng/ml, R&D Systems, Minneapolis, Minn.).

The medium from the differentiated macrophage cell cultures was removedand replaced with similar medium or with medium containing low densitylipoprotein (LDL, Sigma), oxidized LDL (oxidized by incubation in 5 μMCuSO₄.5H₂O according to the method of Malden et al., J. Biol. Chem.,266:13901 (1991)), or dexamethazone (6 nM, Sigma Chemical Co.).Following 3 days of each treatment, the culture medium was removed,brought to pH 6.8 by the addition of HCl, and passed over aHeparin-Sepharose CL-6B column (Pharmacia, Piscataway, N.J.). The columnwas washed with 0.2 M NaCl in 20 mM Tris, pH 8, and eluted with 0.6 MNaCl in 20 mM Tris, pH 8. The eluted material was fractionated on an 18%acrylamide SDS-PAGE gel (NOVEX) and electroblotted to PVDF membrane(Millipore, Bedford Mass.). The filter was blocked, washed, and reactedwith monoclonal antibodies against MDC using standard technique(Sambrook et al.). In each of the culture media analyzed, MDC proteinwas detected at a concentration of approximately 0.5 μg/ml, thusconfirming expression of MDC in differentiated human macrophages.

Expression of MDC also was analyzed in human epithelial cell lines. Thecolon epithelial cell line T84 (ATCC #CCL-248) was grown in DMEM/F12medium (GIBCO, Gaithersburg Md.), and the lung epithelial cell line A549(ATCC #CCL-185) was grown in F12 medium. Screening for the presence ofMDC mRNA in the cells and MDC protein in the culture medium wasperformed as described above for macrophages. No evidence of MDCexpression was detectable by either method in these cell lines.

In addition, samples of the T84 cell line were treated for 1 day withTNFα (5 ng/ml, PeproTech, Rocky Hill, N.J.), TGF-β (1 ng/ml, R&DSystems), or interferon-γ(200 U/ml, PeproTech), each with or withoutaddition of recombinant MDC at 100 ng/ml (derived from CHO celltransfectants; see Ex. 10). Samples of the A549 cell line were treatedwith 50 ng/ml PMA (Sigma Chemical Co.) for 0, 1, 3, 5, or 7 days. Noneof these treatments resulted in detectable expression of MDC mRNA in theT84 or A549 cells when screened by Northern blotting as described above.

C

Further examination of MDC gene expression in macrophages was conductedtreating the human cell line HL60 with either 1% DMSO (Sigma ChemicalCo.) or 50 ng/nml PMA (Sigma). Treatment with DMSO inducesdifferentiation of HL60 cells into a granulocytic cell type, whereas PMAinduces their differentiation into a macrophage lineage [Perussia etal., Blood, 58: 836-843 (1981)]. RNA was isolated from untreated cellsand from cells treated for one three days with DMSO or PMA,electrophoresed (10 μg/lane), and blotted. The Northern blot of the RNAwas probed with the radiolabeled pMP390 5′ fragment described in Example3.

After three days of PMA treatment, the HL-60 cells clearly expressed MDCmRNA, although the level of expression was apparently less than that ofmacrophages after six days of culture (see above). No expression wasseen after one day of treatment or in untreated cells. Further, nodetectable expression of MDC was induced by treatment with DMSO for oneor three days.

EXAMPLE 5 In Situ Hybridization

Because MDC gene expression was detected in the thymus and spleen, insitu hybridization was carried out to localize the source of the messagein these tissues. Further, in situ hybridization was used to correlateMDC gene expression with tissue inflammation, using intestinal tissuefrom Crohn's diseased patients as an example. The procedures used forthese experiments are described in detail in U.S. Ser. No. 08/939,107and WO 96/40923, both of which are incorporated by reference.

Observed hybridization of the anti-sense strand indicated that the MDCgene was expressed in cells throughout the cortex of normal humanthymus, with weak signal in the follicles. Expression of MDC in thethymus may indicate a T lymphocyte developmental role of MDC. Expressionin normal human spleen was localized to cells of the red pulp, whereaslittle signal was detected in the white pulp. A high level of expressionin inflamed tonsil was localized to the epithelial region, althoughinflammatory cells appeared to have infiltrated the entire tissuesample.

Colon samples from patients with Crohn's disease exhibited hybridizationin cells of the epithelium, lamina propria, Payer's patches, and smoothmuscle. In contrast, normal human colon showed no hybridization abovebackground. The observed pattern of ADC expression in the colons ofCrohn's disease patients closely correlates with the expression of amacrophage-specific gene, Platelet Activating Factor Acetylhydrolase(PAF-AH) [Tjoelker et al., supra]. This result, together with the datapresented in Example 4, suggest that macrophages express MDC cDNA invivo during pathogenic inflammation. Moreover, the identification of MDCin Crohn's disease colon tissue samples suggest diagnostic relevance ofMDC levels (e.g., in a patient's blood, stool sample, and/or intestinallesions) to a patient's disease state or clinical prognosis.

EXAMPLE 6 Production of Recombinant MDC

To produce recombinant MDC protein, the sequence encoding the putativemature form of the protein was amplified by PCR and cloned into thevector pGEX-3× (Pharmacia, Piscataway, N.J.). The pGEX vector isdesigned to produce a fusion protein comprisingglutathione-S-transferase (GST), encoded by the vector, and a proteinencoded by a DNA fragment inserted into the vector's cloning site.

An MDC cDNA fragment was amplified by PCR using the primers 390-2R (SEQID NO: 8) and 390-FX2 (SEQ ID NO: 1 r). Primer 390-FX2 contains a BamH Irestriction site followed by a sequence encoding a thrombin cleavagesite [Chang et al., Eur. J. Biochem., 151:217 (1985)] followed by bases92-115 of SEQ ID NO: 1. The thrombin cleavage site is as follows:leucine-valine-proline-arginine-glycine-proline, in which glycine andproline are the first two residues of the mature form of MDC. Treatmentof the recombinant fusion protein with thrombin is expected to cleavethe arginine-glycine bond of the fusion protein, releasing the maturechemokine from the GST fusion.

The PCR product was purified by agarose gel electrophoresis, digestedwith BamH I endonuclease, and cloned into the BamH I site of pGEX-3×.This pGEX-3×/MDC construct was transformed into E. coli XL-1 Blue cells(Stratagene, La Jolla Calif.), and individual transformants wereisolated and grown. Plasmid DNA from individual transformants waspurified and partially sequenced using an automated sequencer and primerGEX5 (SEQ ID NO: 12), which hybridizes to the pGEX-3× vector near theBamHI cloning site. The sequence obtained with this primer confirmed thepresence of the desired MDC insert in the proper orientation.

Induction of the GST-MDC fusion protein was achieved by growing thetransformed XL-1 Blue culture at 37CC in LB medium (supplemented withcarbenicillin) to an optical density at wavelength 600 nm of 0.4,followed by further incubation for 4 hours in the presence of 0.25 to1.0 mM Isopropyl β-D-Thiogalactopyranoside (Sigma Chemical Co., St.Louis Mo.).

The fusion protein, produced as an insoluble inclusion body in thebacteria, was purified as follows. Cells were harvested bycentrifugation; washed in 0.15 M NaCl, 10 mM Ttris pH 8, 1 mM EDTA; andtreated with 0.1 mg/mnl lysozyme (Sigma Chemical Co.) for 15 minutes atroom temperature. The lysate was cleared by sonication, and cell debriswas pelleted by centrifugation for 10 minutes at 12,000×g. The fusionprotein-containing pellet was resuspended in 50 mM Tris, pH 8, and 10 mMEDTA, layered over 50% glycerol, and centrifuged for 30 min. at 6000×g.The pellet was resuspended in standard phosphate buffered salenesolution (PBS) free of Mg⁺⁺ and Ca⁺⁺. The fusion protein, which remainedinsoluble, was approximately 80-90% of the protein mass and migrated indenaturing SDS-polyacrylamide gels with a relative molecular weight of33 kD. The protein yield, as judged by Coomassie staining, wasapproximately 100 mg/l of E. coli culture.

The fusion protein was subjected to thrombin digestion to cleave the GSTfrom the mature MDC protein. The digestion-reaction (20-40 μg fusionprotein, 20-30 units human thrombin (4000 U/mg (Sigma) in 0.5 ml PBS)was incubated 16-48 hrs. at room temperature and loaded on a denaturingSDS-PAGE gel to fractionate the reaction products. The gel was soaked in0.4 M KCl to visualize the GST and MDC protein bands, which migrated asfragments of approximately 26 kD and 7 kD, respectively.

The identity of the 7 kD SDS-PAGE fragment was confirmed by partialamino acid sequence analysis. First, the protein was excised from thegel, electroeluted in 25 mM Tris base and 20 mM glycine, and collectedonto a PVDF membrane in a ProSpin column (Applied Biosystems, FosterCity, Calif.). Subjecting the sample to automated sequencing (AppliedBiosystems Model 473A, Foster City, Calif.) yielded 15 residues ofsequence information, which corresponded exactly to the expectedN-terminus of the predicted mature form of MDC (SEQ ID NO: 2, amino acidresidues 1 to 15).

EXAMPLE 7 Production of Recombinant MDC in Bacteria

MDC peptides and analogs can be expressed using a variety of bacterialexpression systems including E. coli, Bacillus subtilis, streptomyceslividans, and many others. [For a general review see “Gene ExpressionTechnology” in Methods in Enzymology, Vol. 185: pp. 1-283, Ed. D. V.Goeddel, Academic Press, San Diego, Calif. (1990).] In general, anexpression cassette comprised of a transcription element (a promoter), atranslation element, a coding region to be expressed (for example MDC),and a transcription termination-element is developed and optimized toeffect significant gene expression. This cassette is incorporated intoeither episomal plasmids, which confer stable propagation, or intointegration vectors to mediate the insertion or creation (via homologousrecombination) of an expression cassette within the host genome. Thegene can be expressed directly or can be fused to signal sequences(e.g., pelB, ompA, est2) to direct secretion of the gene product out ofthe cytoplasm into either the periplasmic space or media, or to otherleader sequences (e.g., ubiquitin) to enhance the folding or otherwisestabilize the recombinantly expressed coding region. The gene product,either properly folded or not, can be recovered in a crude state or asinclusion bodies from the cells following a fermentation phase andeither directly purified or refolded prior to purification.

A. Construction and Testing of Bacterial MDC Expression Vector P2-390

The portion of the MDC cDNA encoding the predicted mature MDC proteinwas cloned into a plasmid containing the arabinose promoter (araB) andthe pelB leader sequence [see Better et al., Science, 240:1041-43(1988)].

More particularly, an MDC cDNA was amplified by PCR using approximately0.1 μg of pMP39012 as template and synthetic oligonucleotide primers390-2R (SEQ ID NO:8) and 390-Pel (SEQ ID NO: 13). Primer 390-Pelcontains an Nco I restriction site, followed by two cytosine residues,followed by bases 92 to 115 of SEQ ID NO: 1.

The expected PCR product of 232 bp was purified by agarose gelelectrophoresis digested with Nco I and BamH I, and cloned along with aportion of the arabinose operon and peIB leader sequence (Better et al.,supra) into the vector pUC19 (New England Biolabs, Beverly Mass.). Theresultant construct, designated P2-390, encodes a fusion of the pelBleader (encoded by the vector) to the mature MDC protein. The sequenceof this construct was confirmed by automated sequencing using theprimers Ara1 (SEQ ID NO:28) and Ara2 (SEQ ID NO:29) which anneal to thevector adjacent to the cloning site. The plasmid P2-390 was transformedinto the E. coli strain MC1061 using standard procedures, and anampicillin resistant clone was selected for MDC production. The clonewas grown in a 3 liter fermenter (Applikon, Foster City, Calif.) and MDCproduction was induced by the addition of 50% arabinose to a finalconcentration of 0.1%. After one day of cultivation in the presence ofarabinose, the cells were harvested Western blotting revealed that MDCwas present within the cells at a level of approximately 4 μg/g of cellpaste and was secreted into the culture medium to a level ofapproximately 1 μg/m.

B. Protocol for Bacterial Expression of MDC Using Plasmid P2-390

The plasmid P2-390 was transformed into E. coli strain SB7219 (Sheppardand Englesberg, J. Molec. Biol., 25:443454 (1967) and Wilcox et al., J.Biol. Chem., 249:2946-2952 (1974)). SB7219 is a prototrophic strainincapable of degrading arabinose, the inducer of the araB promoter usedto transcribe the peIB-MDC coding region. The genotype of SB7219 is E.coli K12 F⁻ del(codb-lac)3 del(ara735) rpsL150(str^(R))λ⁻. Theproduction strain SB7219:P2-390 was grown in the fermenter (run FC563)in a fed batch format. A frozen aliquot of the seed is inoculated into250 ml of fermentation basal medium in the shake flask. The compositionof the basal medium is as follows: Basal Medium Component Quantity per LNa₃citrate 1 g 5.4% FeCl₃.6H₂0 2 ml glucose 2 g NaH₂PO₄.H₂0 3 g K₂HPO₄ 6g (NH₄)₂SO₄ 5 g 20% yeast extract solution 5 ml 1 M CaCl₂ 0.5 ml 1 MMgCl₂ 2.0 ml trace elements 4 ml trace vitamins 2 ml 1% thiamine 1 mltetracycline 5 mg pH is set to 7.0

Trace Elements Solution Component Quantity per L Boric Acid 5.0 g CopperSulfate.5H₂0 2.0 g Potassium Iodide 1.0 g Manganese sulfate  10 gMolybdic acid 0.5 g ZnCl₂ (Anhydrous) 5.2 g Cobalt chloride 0.5 g

Trace Vitamin Solution Component Quantity per L Sodium Hydroxide, 50%1.3 ml Riboflavin 0.42 g Folic Acid 0.04 g D-Pantothenic Acid(hemicalcium salt) 5.4 g Nicotiinic Acid (niacin) 6.1 g Pyridoxine HCl1.4 g Biotin 0.06 g

The shake flask culture is grown at 37° C. and 220 RPM to an opticaldensity corresponding mid-exponential growth (approximately OD₆₀₀≈0.7).The inoculum is added to the ferment containing 1.5 L of basal media andgrown at 30° C. for 5 hours. A feed is then initiated at 3 ml/hr andexponentially increased to effect a doubling time of 5 hr until amaximum of 18 ml/hr of feed is achieved. Feed Medium Component Quantityper L Na₃citrate 5 g 5.4% FeCl₃.6H₂0 10 ml glycerol 500 g (NH₄)₂SO₄ 5 g1 M CaCl₂ 4 ml 1 M MgCl₂ 100 ml 1 M MnCl₂ 0.4 ml trace elements 10 ml

When the wet cell mass is approximately 100 g/L, 20 ml of 50% arabinosesolution is added to induce expression of MDC. The temperature is raisedto 37° C. and the feed rate is decreased to 12 ml/hr. The fermentationis allowed to continue for approximately 20 more hours, at which timethe cell paste is harvested from the tank and stored frozen at −70° C.The MDC contained in the cell paste is suitable for recovery bymechanical lysis, re-folding, and purification as described below inExample 8.

C. Direct Expression of MDC in E. coli

In a similar way, MDC that is directly expressed (i.e., without a fusedin-frame leader sequence) is engineered into the same vector. Theplasmid pBAR5/MDC/RC is a plasmid identical to P2-390 except for theelimination of the pelB leader sequence. In addition, the first fourteenpercent of the MDC(1-69) coding sequence (amino acid codons 1-6 and8-10) have been modified to change cytosine residues at codon positionthree to either an adenosine or thymidine nucleotide (while preservingthe encoded amino acid). Additionally, a translation initiation codonwas added. Thus, the coding sequence in pBAR5/MDC/RC begins:

-   -   5′ ATG GGA CCA TAT GGA GCA AAT ATG GAA GAT AGT . . . . (SEQ ID        NO: 44)        E. coli strain SB7219 harboring this plasmid is grown in a        fermentor essentially as described above and the MDC that is        produced is similarly recovered.

D. P2-390 Variant Expression Vector

In addition, a derivative of P2-390 pBAR5/PelB/MDC/RC in which the aminoacid codons described above in part C were substituted for the wild-typesequence was created. E. coli SB7219 harboring this plasmid is grown ina fermentor in a comparable fashion and the MDC produced is similarlyrecovered.

EXAMPLE 8 Purification of Recombinant MDC from Bacteria and CultureMedium

The following are experimental protocols for purification of therecombinant MDC produced as described in Example 7.

A. Recovery and Purification of Secreted Recombinant MDC.

The secreted recombinant MDC protein is purified from the bacterialculture media by, e.g., adapting methods previously described for thepurification of recombinantly produced RANTES chemokine [Kuna et al., J.Immunol., 149:636642 (1992)], MGSA chemokine [Horuk et al., J. Biol.Chem. 268:541-46 (1993)], and IP-10 chemokine (expressed in insectcells) [Sarris et al., J. Exp. Med, 178:1127-1132 (1993)].

B. Recovery and Re-folding of MDC Bound in Inclusion Bodies

Methods for recovery of inclusion bodies from E. coli paste has beenwell described [see Lin et al., Biotechniques, 11(6): 748-52 (1991);Myers et al., Prot. Express. Purif., 2: 136-143 (1991); Krueger et al.,BioPharm., pp. 4045 (March, 1989); Marston et al., “Solubilization ofProtein Aggregates,” Methods in Enzymology, M P Deutcher (Ed.), AcademicPress, New York, 182: 264-276 (1990)]. Briefly, MDC is released fromintact cells using a mechanical lysis device (e.g., Mauton-Gaulin). Thecell paste is resuspended (20-30% w/v) in buffer [for example,containing 50 mM Tris HCl, pH 8.0, 1 mM EDTA, 50 mM NaCl, 0.2 mg/mllysoyme, and 0.5% (v/v) Triton X-100] and passed through the machine ata constant pressure of 8-12,000 PSI for one to two passes at 4-15° C.The soluble components of the cell are separated from MDC and the othercellular-derived insoluble components by applying a centrifugal force ofapproximately 12,000×g for a period of about 5-10 minutes. The insolublepelleted material is then re-suspended and re-centrifuged using dilutesolutions of detergent [for example, 0.5% (v/v) Triton X-100 and 10 mMEDTA, pH 8.0]. Other wash steps can be used, including 0.5% (v/v)Zwittergent 3-14 (Calbiochem, Inc.), as well as treatments to minimizeviscosity including lysozyme, DNase, Nonidet and EDTA [seeBartholome-DeBelder et al., Mol. Microbiol., 2:519 (1988)].

To achieve proper folding of MDC contained in exclusion bodies,inclusion body preparations are reduced at a protein concentration of5-10 mg/ml in 6 M guanidine-HCl containing 0.1M Tris HCl, pH 8.6, 20%β-mercaptoethanol, for 1 hour at 37° C. Complete reduction results in acompletely clear solution. Confirmation of complete reduction isobtained using an analytical reverse phase (rp) HPLC procedure. Forexample, a Vydac C4 analytical column (e.g., 214 nin) is equilibrated in5% acetonitrile/water/0.1% trifluoroacetic acid. The sample is injectedand a linear gradient with increasing acetylnitrile content is run at arate of 2% increase per minute. A single peak indicates that completereduction of the MDC protein has been achieved.

The pH of the solution containing the fully reduced MDC is graduallylowered to 4.0 with 10% HCl. The MDC is then recovered from thereduction solution using preparative rpHPLC [e.g., a Vydak C4preparative column with the gradient as described above] to remove HClsalts and denaturant. The recovered MDC is then diluted into 2 Mguanidine-HCl, 0.1 M Tris HCl, pH 8.6, 8 mM cysteine, 1 mM cystine to aprotein concentration of 2 g/L. The solution is stirred slowly at roomtemperature for 4-8 hours and shielded from light. The concentration ofproperly refolded MDC is monitored using the analytical rpHPLC methoddescribed above and is distinguished from reduced MDC by a 2-4 minutereduction in retention time on the HPLC column, relative to the reducedMDC. Confirmation of disulfide bond formation in refolded MDC isconfirmed using mass spectrometry [i.e., MALDI MS].

C. Purification of Refolded MDC

MDC is purified using a two column procedure as follows:SP-Sepharose-fast flow (Pharmacia) resin is packed for columnpurification and equilibrated in loading buffer (0.2 M NaCl, 20 mM Trisbase, pH 7.5). The recovered, refolded MDC solution is diluted withbuffer until the conductivity of the supernatant equals 18-19 mS, andthe pH is adjusted to 7.5. The solution is filtered to remove insolublematerials and applied to the column to a capacity of 0.5 mg MDC/ml ofresin. Loading buffer is then used until the OD₂₈₀ returns to baseline.MDC is eluted using a higher salt buffer (0.6 M NaCl, 20 mM Tris, pH7.5).

The SP-Sephadex elution peak is then chromatographed on an WP Hi-Propyl(C3) hydrophobic interaction column (JT Baker #7585-O₂). The column isequilibrated with 2.4 M NaCl, 20 mM Tris, pH 7.5. The 0.6 M NaClcontaining S—P eluate is then adjusted with the appropriate amount of 5M NaCl to bring the salt concentration of the eluate to 2.4M NaCl. Theadjusted eluate is loaded onto the propyl column at 2 mg of MDC/ml andwashed with 2.4 M NaCl, 20 mM Tris, pH 7.5, until the OD₂₈₀ returns tobaseline. The column is then washed with two column volumes of 2.0 MNaCl, 20 mM NaCl. The purified MDC is eluted from the column with 0.8 MNaCl, 20 mM Tris, pH 7.5. Purified MDC is then filter sterilized andstored at −70° C.

EXAMPLE 9 Recombinant Production of MDC in Yeast

Following are protocols for the recombinant expression of MDC in yeastand for the purification of the recombinant MDC. Heterologous expressionof human genes using microbial hosts can be an effective method toproduce therapeutic proteins both for research and commercialmanufacture. Secretion from yeast hosts (see recent review by Romanos,Yeast, 8: 423-488 (1992)) such as Saccharomyces cerevisiae (Price etal., Gene, 55:287 (1987)) Kluyveromyces lactis (Fleer et al.,Bio/Technology, 9: 968-975 (1991)), Pichia pastoris, (Cregg et al.,Bio/Technology, 11: 905-910 (1993)), Schizosaccharomyces pombe (Brokeret al., FEBS Lett., 248: 105-110 (1989)), and related organisms providea particularly useful approach to obtain both high titer production ofcrude bulk product and rapid recovery and purification. These expressionsystems typically are comprised of an expression cassette containing astrong transcriptional segment of DNA or promoter to effect high levelsof mRNA expression in the host. The mRNA typically encodes a codingregion of interest preceded by an in-frame leader sequence, e.g., S.cerevisiae pre-pro alpha factor (Brake et al., Proc. Nat. Acad. Sci.,81: 4642-4646 (1984)) or equivalent signal, which directs the maturegene product to the culture medium. As taught below, MDC can beexpressed in such a manner.

In one exemplary protocol, the coding region of the MDC cDNA isamplified from pMP390-12 by PCR, using as primers syntheticoligonucleotides containing the MDC cDNA sequences present in primers390-1F (SEQ ID NO: 7) and 390-2R (SEQ ID NO: 8). A DNA encoding theyeast pre-pro-alpha leader sequence is amplified from yeast genomic DNAin a PCR reaction using one primer containing bases 1-20 of the alphamating factor gene and another primer complimentary to bases 255-235 ofthis gene [Kujan and Herskowitz, Cell, 30: 933-943 (1982)]. Thepre-pro-alpha leader coding sequence and MDC coding sequence fragmentsare ligated into a plasmid containing the yeast alcohol dehydrogenase(ADH2) promoter, such that the promoter directs expression of a fusionprotein consisting of the pre-pro-alpha factor fused to the mature MDCpolypeptide. As taught by Rose and Broach, Meth. Enz., 185: 234-279, D.Goeddel, ed., Academic Press, Inc., San Diego, Calif. (1990), the vectorfurther includes an ADH2 transcription terminator downstream of thecloning site, the yeast “2-micron” replication origin, the yeast leu-2dgene, the yeast REP1 and REP2 genes, the E. coli beta-lactamase gene,and an E. coli origin of replication. The beta-lactamase and leu-2dgenes provide for selection in bacteria and yeast, respectively. Theleu-2d gene also facilitates increased copy number of the plasmid inyeast to induce higher levels of expression. The REP1 and REP2 genesencode proteins involved in regulation of the plasmid copy number.

The DNA construct described in the preceding paragraph is transformedinto yeast cells using a known method, e.g., lithium acetate treatment[Stearns et al., Meth. Enz., supra, pp. 280-297]. The ADH2 promoter isinduced upon exhaustion of glucose in the growth media [Price et al.,Gene, 55:287 (1987)]. The pre-pro-alpha sequence effects secretion ofthe fusion protein from the cells. Concomitantly, the yeast KEX2 proteincleaves the pre-pro sequence from the mature MDC chemokine [Bitter et.al., Proc. Natl. Acad. Sci. USA, 81:5330-5334 (1984)].

Alternatively, MDC is recombinantly expressed in yeast using acommercially available expression system, e.g., the Pichia ExpressionSystem (Invitrogen, San Diego, Calif.), following the manufacturer'sinstructions. This system also relies on the pre-pro-alpha sequence todirect secretion, but transcription of the insert is driven by thealcohol oxidase (AOX1) promoter upon induction by methanol.

The secreted MDC is purified from the yeast growth medium by, e.g., themethods used to purify MDC from bacterial and mammalian cellsupernatants (see Examples 8 and 10).

MDC was expressed in yeast as follows. Using standard molecularbiological methods (Sambrook et al., Molecular Cloning: a LaboratoryManual, Second Edition, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1989)) such as those described above, the S. cerevisiaealpha factor pre-pro sequence (codons 1-85 in FIG. 9) was fused to thepresumptive mature form of MDC (SEQ D NO: 1, positions 1-69; codons86-155 in FIG. 9). Expression of the resultant coding region is undercontrol of the K lactis LAC4 promoter present in the plasmidpYGL/preproMC (see FIG. 10). This plasmid is a derivative of the K.lactisexpression plasmid developed by Fleer et al., (supra) and used tosecrete high titers of human serum albumin. This vector class is derivedfrom the plasmid pKD1, a 2μ like plasmid from in K. drosophilarium (Chenet al., Nucleic Acids Research, 14: 447-81 (1986)). These vectors areautonomously replicated and maintained at high copy number and have beenshown to confer high levels of protein production when K lactis strainscontaining these plasmids are grown in either galactose or lactose as“inducing” agents and as the sole carbon source. The constructpYGL/preproMDC confers to the host both resistance to G418 (200 mg/L)and the glycolytic enzyme phosphoglucokinase (PGK). Efficient selectionfor transformed cells containing the plasmid is effected by providing asole carbon source that requires processing via the glycolytic pathwayof intermediary metabolism.

Plasmid pYGL/preproMDC was transformed into the pgko deficient hoststrain FBO5 (Delta Biotechnology Limited) by selecting for G418resistance in YEPPglycerol/ethanol medium (0.5% yeast extract, 1%peptone, 1 M KPO₄, pH 7.0, containing 3% glycerol and 2% ethanol).Following clonal isolation, the transformed seed was grown in shakeflask production medium YEPPgal (0.5% yeast extract, 1% peptone, 1 MKPO4, pH 7.0, containing 2% galactose as sole carbon source). SDS-PAGEanalysis of the culture medium indicated that a protein species of themolecular weight expected of that for mature MDC was present. Thisprotein migrated comparably to synthetic MDC (Gryphon SciencesCorporation). Titration data using dilutions of purified synthetic MDCand culture supernatants in Coomassie blue stained SDS-PAGE gelssuggested that MDC was present in the range of 4-10 mg/L.

Western analyses using an anti-MDC monoclonal antibody did not revealthe presence of MDC-related degradation products, even after furtherculturing of the seed 24 hours past the completion of growth. Thisobservation suggested that the seed is capable of producing and stablyaccumulating MDC, indicating that high cell fermentation methods wouldbe effective to increase titer.

The MDC production seed was used to inoculate a fermentor maintained at26° C. containing a batch medium. The composition of the batch medium(1200 ml) was as follows: 7.5 g Yeast extract; 0.6 g MgSO₄; 6.0 gNH₄SO₄; 9.6 g KH₂PO₄; 26.4 g K₂HPO₄; 11 mg CaCl₂; 5.0 ml 1000× vitamins[Bitter et al., J. Med Virol., 25(2):123-140 (1988)]; 2.5 ml 100× traceelements [Bitter et al. (1988)]; and 1.2 g 30% galactose.

One hour following inoculation, a feed was initiated at a rate of 12ml/hour and maintained for four days. The feed medium composition (1500ml) was as follows: Galactose, 600 g; yeast extract, 50 g; MgSO₄, 4 g;NH₄SO₄, 40 g; KH₂PO₄, 60 g; K₂HPO₄, 165 g; 100× trace elements, 15 ml;1000× vitamins, 30 ml; 4% CaCl₂ solution, 20 ml.

Samples were collected and analyzed throughout the run. MDC accumulatedduring the first three days of the fermentation to a final titer ofapproximately 50 mg/L as determined from purification recoveryexperiments. The primary protein species present is MDC. Significantlevels of degradation were not observed by SDS-PAGE analysis. A sampleof the harvest supernatant was partially purified using ion exchangechromatography. Following dialysis into phosphate buffered saline, theyeast-produced MDC exhibited a single molecular mass of 8088 daltons, ascompared with the theoretical value of 8086, well within the expectederror of the measurement.

Yeast-produced MDC was further analyzed for biological activity bycalcium flux assay and found to exhibit activity comparable to theactivity of synthetic MDC and CHO-produced MDC. Using the assaydescribed below in Example 25, yeast-produced MDC was also successful incompeting with synthetic MDC-SEAP for binding to CCR4 recombinantlyexpressed on a mammalian cell surface.

EXAMPLE 10 Recombinant Production of MDC in Mammalian Cells

MDC was recombinantly produced in mammalian cells according to thefollowing procedures.

A. Synthesis of Expression Vector 390HXE

A truncated version of the MDC cDNA was synthesized by PCR usingpMP390-12 as template and the synthetic oligonucleotides 390RcH (SEQ IDNO: 14) and 390RcX (SEQ ID NO: 15) as primers. Primer 390RcH contains aHind III restriction site followed by bases 1 to 20 of SEQ ID NO: 1;primer 390RcX contains an Xba I restriction site followed by thesequence complimentary to bases 403 to 385 of SEQ ID NO: 1.

The expected 423 bp PCR product was purified by agarose gelelectrophoresis and cloned into Hind III/Xba I-digested pRc/CMV((InVitrogen, San Diego Calif.) a vector which allows for directexpression in mammalian cells). The resulting plasmid, designated390HXE, contained bases 1 to 403 of SEQ ID NO: 1. The sequence of theinsert was confirmed by automated sequencing using the primers DC03 (SEQID NO: 16) and JHSP6 (SEQ ED NO: 3). Primer DC03 anneals to the pRc/CMVvector sequence adjacent to the cloning site.

B. Synthesis of Expression Vector 390HmX

Another MDC cDNA construct was generated by PCR, using pMP390-12 astemplate and the primers 390RcH (SEQ ID NO: 14) and 390mycRX (SEQ ID NO:17). Primer 390mycRX contains an Xba I restriction site, a sequencecomplementary to the sequence encoding a “myc” epitope [Fowlkes et al.,BioTechniques, 13:422427 (1992)], and a sequence complementary to bases298 to 278 of SEQ ID NO: 1. This reaction amplified the expected 354 bpfragment containing bases 1 to 298 of SEQ ID NO: 1 fused to a “myc”epitope at the MDC carboxy-terminus. This epitope can be used tofacilitate immunoprecipitation, affinity purification and detection ofthe MDC-myc fusion protein by Western blotting. The fragment was clonedinto pRc/CMV to generate the plasmid 390HmX. The sequence of the insertwas confirmed by automated sequencing using the primer DC03 (SEQ ID NO:16).

C. Expression of MDC in 293T and NS0 Cells

Two transfection protocols were used to express the two MDC cDNAconstructs described above in subparts A. and B.: transient transfectioninto the human embryonic kidney cell line 293T and stable transfectioninto the mouse myeloma cell line NS0 (ECACC 85110503).

Transient transfection of 293T cells was carried out by the calciumphosphate precipitation protocol of Chen and Okayama, BioTechniques,6:632-638 (1988) and Mol. Cel. Biol., 87:2745-2752 (1987). Cells andsupernatants were harvested four days after transfection. A Northernblot was prepared from 4 μg of total RNA from each cell lysate andprobed with a radiolabeled MDC fragment prepared by PCR The template forthe labeling reaction was a PCR fragment previously generated byamplifying pMP390 with the primers 390-1F (SEQ ID NO: 17) and 390-4R(SEQ ID NO: 9). Approximately 30 ng of this fragment was employed in aPCR reaction containing the following: 1.5 mM MgCl₂, 50 mM KCl, 10 mMTris, pH 8.4, 0.2 mM dATP, 0.2 mM dTTP, 0.2 mM dGTP, 1 μM dCTP, 50 μCiα³²P-dCTP (DuPont/New England Nuclear, Boston Mass.), 2.5 U Taqpolymerase, and 10 μg/ml each of primers 390-1F and 390-2R. The reactionwas denatured by heating for 4 minutes at 94° C., followed by 15 cyclesof amplification (denaturation for 15 seconds at 94° C., annealing for15 seconds at 60° C., and extension for 30 seconds at 72° C.). The probewas purified by passage over a G-25 Quick Spin column (BMB). Conditionsfor hybridization were as follows: The filters were incubated at 42° C.for 16 hours with 5×10⁷ counts per minute (cpm) of the probe, in 40-50ml of a solution containing 50% formamide, 5× Denhardt's solution, 5×SSC(1×SSC is 0.15 M NaCl, 15 mM sodium citrate), 50 mM sodium phosphate, pH6.5, and 0.1 mg/ml sheared salmon sperm DNA (Sigma, St. Louis Mo.).

Filters were subsequently washed in 0.5×SSC and 0.2% SDS at 42° C. for30 minutes. Autoradiography was carried out at −80° C. with oneintensifying screen for sixteen hours. The MDC DNA constructs were veryhighly expressed in the transfected cells and not detectable in thenon-transfected cells.

For stable transfections, NS0 cells were grown to 80% confluency inD-MEM (Gibco), collected by centrifugation, and washed with PBS. Twentyμg of plasmid DNA was linearized with Sca I restriction endonuclease(BMB), added to the cells, and incubated on ice for 15 minutes in a 0.4cm gap cuvette (BioRad, Hercules Calif.). The cells were electroporatedwith two pulses of 3 microfarad at 1.5 kilovolts. Cells were dilutedinto 20 ml D-MEM, incubated at 37° C. in 5% CO₂ for 24 hours, andselected by plating into 96-well plates at various dilutions in D-MEMcontaining 800 μg/ml geneticin. Wells containing single drug-resistantcolonies were expanded in selective media. Total RNA was analyzed byNorthern blotting as described in the preceding paragraph. Message forMDC was seen only in transfected cell lines.

MDC is purified from mammalian culture supernatants by, e.g., adaptingmethods described for the purification of recombinant TCA3 chemokine[Wilson et al., J. Immunol., 145:2745-2750 (1990], or as described belowin subpart F.

D. Expression of MDC in CHO Cells

PCR was used to amplify bases 1 to 403 of the MDC cDNA clone (SEQ IDNO: 1) using primers 390RcH and 390RcX (SEQ. ID NOs: 14 and 15), asdescribed above in subpart A. The fragment was cloned into the HindIIIand XbaI sites of the expression vector pDC1, a pUC19 derivative thatcontains the cytomegalovirus (CMV) promoter to drive expression of theinsert. More specifically, vector pDC1, depicted in FIG. 8, was derivedfrom pRc/CMV and pSV2-dhfr (ATCC vector #37146). Vector pDC1 is similarto the mammalian expression vector pRc/CMV (Invitrogen, San Diego)except that pDC1 carries the mouse dihydrofolate reductase (dhfr) geneas a selectable marker, in place of the neomycin phosphotransferasegene. Transcription of the target gene in pDC1 is under the control ofthe strong CMV promoter. See Stenberg et at, J. Virology, 49:190-199(1984). Additionally, a polyadenylation sequence from the bovine growthhormone gene [Goodwin and Rottman, J. Biol. Chem., 267:16330-16334(1992)] is provided on the 3′ side of the target gene. The dhfrexpression cassette [Subramani et al., Mol. Cell. Biol. 1:854-864(1981)] allows selection for pDC1 in cells lacking a functional dhfrgene.

XL-1 Blue bacteria (Stratagene) were transformed with the pDC1/MDCplasmid using standard techniques of CaCl₂ incubation and heat shock(Sambrook et al.). Transformants were grown in LB medium containing 100μg/ml carbenicillin. Plasmid DNA from individual transformed clones wasisolated using the Promega Wizard Maxiprep system (Madison, Wis.) andits sequence was confirmed by automated sequencing using the primers390-IF and 390-2R (SEQ ID NOs: 7 & 8). The plasmid was linearized byrestriction digestion with Pvu I endonuclease (Boehringer Mannheim),which cuts once within the vector sequence.

The Chinese hamster ovary (CHO) cell line used for production of MDC wasDG-44, which was derived by deleting the dhfr gene. See Urlaub et al.,Cell, 33:405 (1983). For electroporation, 10⁷ of these CHO cells werewashed in PBS, resuspended in 1 ml PBS, mixed with 25 μg of linearizedplasmid, and transferred to a 0.4 cm cuvette. The suspension waselectroporated with a Biorad Gene Pulser (Richmond, Calif.) at 290volts, 960 μFarad. Transfectants were selected by growth in α⁻ medium(Cat. No. 12000, Gibco, Gaithersburg, MD) containing 10% dialyzed fetalbovine serum (FBS) (Hyclone, Logan, Utah) and lacking hypoxanthine andthymidine. Cells from several hundred transfected colonies were pooledand re-plated in α⁻ medium containing 20 nM methotrexate (Sigma, St.Louis, Mo.). Colonies surviving this round of selection were isolatedand expanded in α⁻ medium containing 20 nM methotrexate.

E. Purification of MDC for Protein Sequencing

Transfected CHO clones were grown on plastic tissue culture dishes toapproximately 90% confluence in α⁻ medium, at which time the medium wasreplaced with P5 medium containing 0.2% to 1.0% FBS. P5 medium consistsof the components listed in Table 2, below (purchased as a premixedpowder form Hyclone, Logan Utah), supplemented with the followingadditional components: (1) 3 g/l sodium bicarbonate (Sigma, St. Louis,Mo.); (2) 2 μg/l sodium selenite (Sigma); (3) 1% soy bean hydrolysate(Quest International, Naarden, The Netherlands); (4) 1× ferroussulfate/EDTA solution (Sigma); (5) 1.45 ml/l EX-CYTE VLE solution(Bayer, Kankakee, Ill.); (6) 10 μg/ml recombinant insulin (Nucellin, EliLily, Indianapolis, Ind.); (7) 0.1% pluronic F-68 (Sigma); (8) 30 μg/mlglycine (Sigma); (9) 50 μM ethanolamine (Sigma); and (10) 1 mM sodiumpyruvate (Sigma). TABLE 2 Powder Component #5 gm/L INORGANIC SodiumChloride 4.0 SALTS Potassium Chloride 0.4 Sodium Phosphate Dibasic,Anhydrous 0.07102 Sodium Phosphate Monobasic H₂0 0.0625 MagnesiumSulfate, Anhydrous 0.1 Cupric sulfate 5H₂0 0.00000125 Ferrous Sulfate7H₂0 0.000417 Zinc Sulfate 7H₂0 0.0004315 Ferric Nitrate 9H₂0 0.00005Calcium Chloride, Anhydrous 0.11661 Magnesium Chloride, Anhydrous 0AMINO ACIDS L-Alanine 0 L-Arginine HCl 0.15 L-Asparagine H₂0 0.075L-Aspartic Acid 0.04 L-Cysteine HClH₂0 0.035 L-Cystine 2HCl 0.12L-Glutamic Acid 0.02 L-Glutamine 0.5846 Glycine 0.02 L-Histidine HClH₂00.04 L-Isoleucine 0.15 L-Leucine 0.15 L-Lysine HCl 0.1 L-Methionine 0.05L-Proline 0.05 L-Phenylainine 0.05 L-Serine 0.075 L-Threonine 0.075L-Tryptophan 0.02 L-Tyrosine 2Na2H₂0 0.075 L-Valine 0.125 VITAMINSBiotin 0.001 D-Calcium Pantothenate 0.0025 Choline Chloride 0.015 FolicAcid 0.005 i-Inositol 0.175 Nicotinamide 0.005 Pyridoxal HCl 0.005Pyrdoxine HCl 0.005 Riboflavin 0.001 Thiamine HCl 0.005 Cyanocobalamine0.001 OTHER D-Glucose 1.0 Hypoxanthine, Na 0.005 Thymidine 0.005Putrescine 2HCl 0.000081 Sodium Pyruvate 0.11004 Linoleic Acid 0.0001DL-Alpha-Lipoic Acid 0.0002 Phenol Red, Na Salt 0.0086022

After two additional days in culture, an aliquot of each supernatant wasmixed with an equal volume of acetone. The precipitated proteins werepelleted by centrifugation, fractionated on an 18% Tris Glycine gel(NOVEX), and blotted to a PVDF membrane (Millipore, Bedford, Mass.).

MDC bound to the membrane was detected by a crude preparation ofmonoclonal antibody to MDC (prepared as described in Example 18). Cellsfrom the clone secreting the highest level of MDC protein (approx. 1μg/ml) were removed from the plate by treatment with a solution of 0.5%trypsin and 5.3 mM EDTA (GIBCO) and used to start a suspension culturein a medium plus 10% fetal bovine serum (FBS). Over the course of 8days, 5 volumes of P5 medium were added to the culture. Proteins wereprecipitated from the culture supernatant by addition of polyethyleneglycol (MW 8000, Union Carbide, Danbury, Conn.) to 20% (weight/volume),fractionated on an 18% Tris glycine gel, and electroblotted to a PVDFmembrane (Millipore, Bedford, Mass.) in CAPS buffer(3-[Cyclohexylamino]-1-propanesulfonic acid, pH 10.4) (Sigma, St. Louis,Mo.). A strip of the filter was removed for detection of MDC by westernblotting with the supernatant from a hybridoma cell line producinganti-MDC monoclonal antibodies (See Example 18). The reactive band,which migrated with an apparent molecular weight of 6.4 kD, was excisedfrom the remaining portion of the filter.

Using an automated sequencer (Applied Biosystems, Model 473A, FosterCity CA), the sequence of the N-terminus of the protein was determinedto be: GPYGANMEDS. This sequence is identical to that of residues 1 to10 of SEQ D NO. 2, corresponding to the N-terminus of the predictedmature form of MDC.

F. Purification of MDC for Biological Assays

For growth of larger cultures, MDC-expressing CHO cells were grown to80% confluence on tissue culture plates in α⁻ medium. The cells wereremoved from the plates by treatment with trypsin and EDTA andresuspended at a density of 3×10⁵ cells/ml in P5 medium plus 1% FBS in aspinner flask at 37° C. Additional P5/1% FBS medium was added as neededto keep the cell density in the range of 1×10⁶ to 3×10⁶.

After 11 days in culture, the cells were removed from the medium byfiltration. The pH of the culture medium was adjusted to 6.8, and it waspassed over a heparin-Sepaharose CL-6B column (Pharmacia, Piscataway,N.J.). After washing with 0.2 M NaCl in potassium phosphate buffer, pH7, the column was eluted with a linear gradient of 0.2 to 0.7 M NaCl.Fractions were analyzed by SDS-PAGE and Coomassie stained to determinewhich of them contained MDC. MDC eluted from the column at approximately0.6 M NaCl.

The fractions containing MDC were pooled and concentrated byultrafiltration in stirred-cell chamber (Amicon, Beverly, Mass.) using afilter with a MW cutoff of 3 kD. Octylglucoside (10 mM finalconcentration, Boehringer Mannheim Biochemicals) was added to theconcentrated MDC, which subsequently was passed through a SephacrylHR100 column (Pharmacia, Piscataway, N.J.). Fractions were analyzed bySDS-PAGE for the presence of MDC. The final yield of MDC protein wasapproximately 0.1 mg/liter of culture supernatant, and the purity wasestimated to be greater than 95%, as judged by Coomassie staining.

EXAMPLE 11 Production of MDC and MDC Analogs by Peptide Synthesis

MDC and MDC polypeptide analogs are prepared by chemical peptidesynthesis using techniques that have been used successfully for theproduction of other chemokines such as IL-8 [Clark-Lewis et al., J.Biol. Chem., 266:23128-34 (1991)] and MCP-1. Such methods areadvantageous because they are rapid, reliable for short sequences suchas chemokines, and enable the selective introduction of novel, unnaturalamino acids and other chemical modifications.

For example, MDC and MDC analogs were chemically synthesized usingoptimized stepwise solid-phase methods [Schnolzer et al., Int. J. Pept.Protein Res., 40:180 (1992)] based on t-butyloxycarbonyl (Boc)chemistries of Merrifield [J. Am. Chem. Soc., 85:2149-2154 (1963)] on anApplied Biosystems 430A Peptide Synthesizer Roster City, Calif.). Theproteins were purified by reverse-phase HPLC and characterized bystandard methods, including electrospray mass spectrometry and nuclearmagnetic resonance.

The chemically synthesized MDC corresponded to the mature form ofrecombinant MDC, consisting of residues 1 to 69 of SEQ ID NO. 2. Severalmethods were used to compare the chemically synthesized MDC to therecombinant MDC produced by CHO cell transfectants as described inExample 10. The migration of chemically synthesized MDC was identical tothat of the recombinant MDC in denaturing SDS-PAGE (18% Tris glycinegel, NOVEX). In addition, the proteins reacted similarly in western blotanalysis using monoclonal and polyclonal antibodies raised againstbacterially produced MDC as described below in Example 18. Thechemically synthesized MDC also appeared to behave in the same manner asthe recombinant MDC in immunoprecipitation assays with the anti-MDCmonoclonal antibodies. These studies indicate that the denatured and thenon-denatured structures of chemically synthesized MDC are similar tothose of recombinant MDC.

The following MDC analogs also have been chemically synthesized:

-   1. “MDC (n+1)” (SEQ ID NO: 30) consists of Leucine followed by    residues 1 to 69 of SEQ ID NO. 2. This analog has alternatively been    referred to herein as “MDC(0-69).”-   2. “MDC (9-69)” consists of residues 9 to 69 of SEQ ID NO. 2.-   3. “MDC-yl” (SEQ ID NO: 31) consists of residues 1 to 69 of SEQ D    NO. 2, with the following substitution: Residues 59-60 (Trp-Val)    were replaced with the sequence Tyr-Leu. A related analog “MDC-wvas”    consists of residues 1 to 69 of SEQ ID NO. 2, with the following    substitution: Residues 59-60 (Trp-Val) were replaced with the    sequence Ala-Ser.-   4. “MDC-eyfy” (SEQ ED NO: 32) consists of residues 1 to 69 of SEQ ID    NO. 2, with the following substitution: Residues 28-31    (His-Phe-Tyr-Trp) were replaced with the sequence Glu-Tyr-Phe-Tyr,    derived from the amino acid sequence of the chemokine RANTES    (residues 26-29 of SEQ ID NO: 21).

The analogs “MDC (n+1)”, “MDC (9-69)”, and “MDC-yl” are expected to beantagonists of MDC activity, inhibiting MDC activity by competitivelybinding to the same receptor that recognizes MDC. Alternatively, theymay effect inhibition by forming inactive heterodimers with the nativeMDC. Possible activities of the analog “MDC-eyfy” include inhibition ofMDC as described for the previous analogs. Alternatively, “MDC-eyfy” mayconfer some of the activities typical of the chemokine RANTES, such aschemotaxis of T lymphocytes, monocytes, or eosinophils.

Additionally, the following single-amino acid alterations (alone or incombination) are specifically contemplated: (1) substitution of anon-basic amino acid for the basic arginine and/or lysine amino acids atpositions 24 and 27, respectively, of SEQ ID NO: 2; (2) substitution ofa charged or polar amino acid (e.g., serine, lysine, arginine,histidine, aspartate, glutamate, asparagine, glutamine or cysteine) forthe tyrosine amino acid at position 30 of SEQ ID NO: 2, the tryptophanamino acid at position 59 of SEQ ID NO: 2, and/or the valine amino acidat position 60 of SEQ ID NO: 2; and (3) substitution of a basic orsmall, non-charged amino acid (e.g., lysine, arginine, histidine,glycine, alanine) for the glutamic acid amino acid at position 50 of SEQID NO: 2. Specific analogs having these amino acid alterations areencompassed by the following formula (SEQ ID NO: 25): Met Ala Arg LeuGln Thr Ala Leu Leu Val Val Leu Val Leu Leu Ala−24             −20                 −15                 −10 Val Ala LeuGln Ala Thr Glu Ala Gly Pro Tyr Gly Ala Asn Met Glu             −5                   1               5 Asp Ser Val Cys CysArg Asp Tyr Val Arg Tyr Arg Leu Pro Leu Xaa     10                  15                  20 Val Val Xaa His Phe XaaTrp Thr Ser Asp Ser Cys Pro Arg Pro Gly 25                  30                  35                  40 Val ValLeu Leu Thr Phe Arg Asp Lys Xaa Ile Cys Ala Asp Pro Arg                 45                  50                  55 Val Pro XaaXaa Lys Met Ile Leu Asn Lys Leu Ser Gln             60                  65wherein the amino acid at position 24 is selected from the groupconsisting of arginine, glycine, alanine, valine, leucine, isoleucine,proline, serine, threonine, phenylalanine, tyrosine, tryptophan,aspartate, glutamate, asparagine, glutamine, cysteine, and methionine;wherein the amino acid at position 27 is independently selected from thegroup consisting of lysine, glycine, alanine, valine, leucine,isoleucine, proline, serine, threonine, phenylalanine, tyrosine,tryptophan, aspartate, glutamate, asparagine, glutamine, cysteine, andmethionine; wherein the amino acid at position 30 is independentlyselected from the group consisting of tyrosine, serine, lysine,arginine, histidine, aspartate, glutamate, asparagine, glutamine, andcysteine, wherein the amino acid at position 50 is independentlyselected from the group consisting of glutamic acid, lysine, arginine,histidine, glycine, and alanine; wherein the amino acid at position 59is independently selected from the group consisting of tryptophan,serine, lysine, arginine, histidine, aspartate, glutamate, asparagine,glutamine, and cysteine; and wherein the amino acid at position 60 isindependently selected from the group consisting of valine, serine,lysine, arginine, histidine, aspartate, glutamate, asparagine,glutamine, and cysteine. Such MDC polypeptide analogs are specificallycontemplated to modulate the binding characteristics of MDC to chemokinereceptors and/or other molecules (e.g., heparin, glycosaminoglycans,erythrocyte chemokine receptors) that are considered to be important inpresenting MDC to its receptor.

Additionally, analogs wherein the proline at position 2 of SEQ ID NO: 1is deleted or substituted for by another amino acid are specificallycontemplated. Such mutants will collectively be referred to as “MDCΔPro₂polypeptides.” As described below in Example 20, MDC (3-69) derived froman HIV-infected T cell line displays properties that are, at least insome respects, opposite or antagonistic from properties observed formature MDC (1-69). It is hypothesized that a dipeptidyl amino peptidasesuch as CD26 [Oravecz el al., J. Exper. Med., 186:1865 (1997)] possessesa specificity for the sequence NH₂-X-Pro (wherein X is any amino acid),and that the dipeptidase therefore is capable of converting mature MDC(1-69) (having the amino terminus NH₂-Gly-Pro-Tyr) to the MDC (3-69)form in vivo. It is expected that the dipeptidase CD26 will not cleavethe amino terminus from MDCΔPro₂ polypeptides, rendering such mutantsmore stable than MDC(1-69) in vivo. MDCΔPro₂ polypeptides that retainthe biological activities of mature MDC (1-69) are useful in alltherapeutic indications wherein MDC (1-69) is useful as a therapeutic,whereas MDCΔPro₂ polypeptides that antagonize the activity of mature MDC(1-69) (e.g., by competitively binding but failing to signal throughCCR4) are useful as MDC antagonists. In preferred embodiments,substitution of the proline with a glycine, alanine, valine, leucine,isoleucine, serine, threonine, phenylalanine, tyrosine, or tryptophan iscontemplated. Introducing the MDCΔPro₂ mutation into any of the analogsdescribed above is also specifically contemplated.

After synthesis, synthetic MDC or MDC analogs may be reduced andrefolded substantially as described in Example 8 forbacterially-produced MDC bound in inclusion bodies, or using proceduresthat are well-known in the art. See, e.g., Protein Folding, T. E.Creighton (Ed.), W.H. Freeman & Co., New York, N.Y. (1992): vanKimmenade et al., Eur. J. Biochem., 173: 109-114 (1988); and PCTpublication no. WO 89/01046.

Recombinant techniques such as those described in the preceding examplesalso are contemplated for preparing MDC polypeptide analogs. Moreparticularly, polynucleotides encoding MDC are modified to encodepolypeptide analogs of interest using well-known techniques, e.g.,site-directed mutagenesis and the polymerase chain reaction. Seegenerally Sambrook et al., supra, Chapter 15. The modifiedpolynucleotides are expressed recombinantly, and the recombinant MDCpolypeptide analogs are purified, as described in the precedingexamples.

The chemoattractant and/or cell-activation properties of MDC or MDCpolypeptide analogs on one or more types of cells involved in theinflammatory process (e.g., T lymphocytes, monocytes, macrophages,basophils, eosinophils, neutrophils, mast cells, and natural killercells), on endothelial cells, epithelial cells, fibroblasts, or othersare assayed by art-recognized techniques that have been used fornumerous other chemokines. Native MDC, recombinant MDC or MDCpolypeptide analogs, or synthetic MDC or MDC polypeptide analogspurified and isolated as described in one or more of the precedingexamples are assayed for activity as described in the following exampleswith respect to MDC.

EXAMPLE 12 Assay of MDC Effects Upon Basophils, Mast Cells, andEosinophils

The effect of MDC upon basophils, mast cells, and eosinophils isassayed, e.g., by methods described by Weber et al., J. Immunol.,154:41664172 (1995) for the assay of MCP-1/2/3 activities. In thesemethods, changes in free cytosolic calcium and release ofproinflammatory mediators (such as histamine and leukotriene) aremeasured. Blocking chemokine-mediated activation of these cell types hasimplications in the treatment of late-phase allergic reactions, in whichsecretion of proinflammatory mediators plays a significant role [Weberet al., supra].

In one signaling assay, synthetic MDC (0.01-10 nM) caused dose-dependentchemotaxis of purified human eosinophils (maximum chemotaxisapproximately four-fold greater than in controls). The relativechemotactic activity of MDC, in relation to other known chemotacticfactors of eosinophils, was as follows:MDC≈eotaxin<RANTES<MCP-4≦eotaxin-2. Eotaxin-2 and MCP-4 were especiallypotent, whereas RANTES effects were intermediate, about one log lesspotent than MCP-4 or eotaxin-2. MDC induced eosinophil migration andshape change even though it did not elicit measurable cytosolic calciumelevations in the eosinophils during these responses. In contrast, theMDC analog MDC(9-69) displayed no chemotactic activity in the sameassay. This data demonstrates a biological activity and utility for MDCin stimulating the chemotaxis of eosinophils, and further demonstrates autility of MDC modulators for modulating this chemotactic activity.

In reported studies with human eosinophils, CCR3 was identified as acritical receptor for a variety of CC chemokines that exert effects oneosinophils, including eotaxin, RANTES, MCP-4 and eotaxin-2. See, e.g.,Garcia-Zepeda, et al., J. Immunol. 157:5613 (1996); Forssman et al., J.Exp. Med, 185:2171 (1997); Stellato et al., J. Clin. Invest., 99:926(1997); and White et al., J. Leukoc. Biol. 62:667 (1997). Also, asreported elsewhere herein, the chemokine MDC binds and signals throughthe chemokine receptor CCR4. However, it was determined that theeosinophil-chemotactic activity of MDC appears to operate in a mannerindependent of the chemokine receptors CCR3 and CCR4. CCR3-transfectedHEK cells labeled with Fura-2 demonstrated a rapid rise in intracellularfree calcium following stimulation with 10-50 nM eotaxin, eotaxin-2, orMCP-4, but not with 10-100 nM MDC. Similarly, purified eosinophilscultured for 72 hours in 10 ng/ml IL-5 and labeled with Fura-2demonstrated a rapid rise in intracellular free calcium followingstimulation with 10-50 nM eotaxin, eotaxin-2, or MCP-4, whereas no suchrise was observed following stimulation with MDC (up to 100 nM). Inaddition, a CCR3 blocking monoclonal antibody was found to inhibiteotaxin- and eotaxin-2-induced chemotaxis of eosinophils, but notchemotaxis induced by MDC.

Two lines of evidence suggest that MDC-induced chemotaxis of eosinophilsoperates independently of CCR4. First, eosinophil cDNA (generated fromeosinophil RNA using oligo-dT or random primers) was screened via PCR.CCR4 could not be detected in either the oligo-dT or random primed cDNA,even though the same PCR primers amplified CCR4 from genomic DNA, andeven though CCR3 nRNA was readily amplifyable. Thus, it appears thateosinophils do not express CCR4. Second, chemotaxis experiments withTARC, a chemokine known to signal through CCR4, have failed (atconcentrations up to 100 nM) to induce chemotaxis of eosinophils.

The fact that MDC apparently exerts its effects on eosinophils in aCCR4-independent manner indicates that, when selecting MDC modulators totreat allergic reactions in which eosinophils play a role, modulatorsthat will have fewer side-effects are those that modulate MDC-inducedchemotaxis of eosinophils without modulating MDC's signaling throughCCR4 Assays are provided herein to select such modulators.

EXAMPLE 13 Assay of Chemoattractant and Cell-Activation Properties ofMDC Upon Human Monocytes/Macrophages and Human Neutrophils

The effects of MDC upon human monocytes/macrophages or human neutrophilsis evaluated, e.g., by methods described by Devi et al., J. Immunol.,153:5376-5383 (1995) for evaluating murine TCA3-induced activation ofneutrophils and macrophages. Indices of activation measured in suchstudies include increased adhesion to fibrinogen due to integrinactivation, chemotaxis, induction of reactive nitrogen intermediates,respiratory burst (superoxide and hydrogen peroxide production), andexocytosis of lysozyme and elastase in the presence of cytochalasin B.As discussed by Devi et al., these activities correlate to severalstages of the leukocyte response to inflammation. This leukocyteresponse, reviewed by Springer, Cell, 76:301-314 (1994), involvesadherence of leukocytes to endothelial cells of blood vessels, migrationthrough the endothelial layer, chemotaxis toward a source of chemokines,and site-specific release of inflammatory mediators. The involvement ofMDC at any one of these stages provides an important target for clinicalintervention, for modulating the inflammatory response.

In one art-recognized chemotaxis assay, a modified Boyden chamber assay,leukocytes to be tested are fluorescently labeled with calcein byincubating for 20 minutes at room temperature. The labeled cells arewashed twice with serum-free RPMI, resuspended in RPMI containing 2mg/ml of BSA, and then added quantitatively to the upper wells of thechambers, which are separated from the lower wells by a polycarbonatefilter (Neuroprobe Inc. Cabin John, Md.). MDC diluted in the same mediumas the leukocytes is added to the lower wells at various concentrations.Chambers are incubated for 2 hours at 37° C. At the end of the assay,cells that have not migrated through the membrane are removed by rinsingthe filter with PBS and scraping with a rubber policeman. Cells thathave migrated through the filter are quantitated by reading fluorescenceper well in a fluorescent plate reader (Cytofluor, Millipore Inc.,Boston, Mass.).

A series of experiments were performed using art-recognized proceduresto determine the chemotactic properties of MDC. Initially, the responseof human mononuclear cells to MDC was determined. The effect of MDC onthe chemotactic response of polymorphonuclear leukocytes (granulocytes)also was examined.

It has been established that MCP-1, which is a C-C chemokine, causesboth recruitment and activation of monocytes but appears to have limitedability to induce the migration of macrophages. The failure of MCP-1 toattract macrophages appears to be correlated to the differentiationprocess: as monocytic cells differentiate, there is a progressivedecrease ir cell response to MCP-1 [Denholm and Stankus, Cytokine, 7:436-440 (1995)]. The biological activities of MCP-1 appear to correlatewith the expression of this chemokine, with MCP-1

mRNA being found in monocytes but decreasing as these cellsdifferentiate.

The pattern of expression of MDC appears to be the reverse of thatdescribed fo

MCP-1, with the amount of mRNA for MDC increasing as monocytesdifferentiate the macrophages. To determine whether this expressionpattern correlates to the biological response to MDC, the effects of MDCon the migration of monocytes and macrophages were compared

A number of different leukocyte cells types were analyzed in chemotaxisan

chemotaxis inhibition assays. Human mononuclear and polymorphonuclearleukocytes wer

isolated from peripheral blood using methods known in the art [Denholmet al., Amer. J Pathol. 135:571-580 (1989)]. Second, the human monocyticcell line, TBP-1 (obtained from the ATCC Rockville, Md., and maintainedin culture in RPM with 10% FBS and wit

pennicillin/steptomycin) was employed. THP-1 cells can be cultured asmonocytes or can b

induced to differentiate to macrophages by treatment with phorbolmyristate acetate (PMA

[Denholm and Stankus, Cytokine, 7:436-440 (1995)]. In some experimentsmonocytic THP-cells were employed, and in others monocytic THP-1 cellswere differentiated to macrophages b

incubation with phorbol myristate acetate (PMA). Third, guinea pigperitoneal macrophages wer

obtained essentially as described in Yoshimura, J. Immunol.,150:5025-5032 (1993). Briefly

animals were given an intraperitoneal injection of 3% sterilethioglycollate (DIFCO) two day

prior to cell harvest. Macrophages were obtained from the peritonealcavity by lavage wit

phosphate buffered saline (PBS) with 1 mM EDTA and 0.1% glucose. Cellswere washed on

by centrifugation and then utilized in chemotaxis assays as describedbelow.

Assays of chemotactic activity were carried out, using the cellpreparation described above, essentially as described by Denholm andStankus, Cytometry, 19:366-3

(1995), using 96-well chambers (Neuroprobe Inc., Cabin John, Md.) andcells labeled with the fluorescent dye, calcein (Molecular Probes,Eugene, Oreg.). Polycarbonate filters used in this ass

were PVP-free (Neuroprobe Inc.); filter pore sizes used for differentcell types were: 5 μm f

monocytes and THP-1 cells, 3 μm for polymorphonuclear leukocytes, and 8μm for guinea pig macrophages.

Fifty thousand calcein labelled cells were resuspended in RPMI mediumcontaining 2 mg/ml BSA and placed in the upper wells. MDC or other testsubstances were diluted in RPMI with BSA (e.g., final MDC concentrationsof 25, 50, 100, 250 ng/ml) and placed in the lower wells. Followingincubation at 37° C. for 2 hours, unmigrated cells remaining above thefilter were removed by wiping; the filter was then air-dried. Controlsin these assays were: RPMI with BSA as the negative control, and 50ng/ml of MCP-1 and 1% zymosan activated serum (ZAS, prepared asdescribed [Denholm and Lewis, Amer. J Pathol., 126:464-474, (1987)])were used as positive controls. Migration of cells was quantitated on afluorescent plate reader (Cytofluor, Millipore Inc. Bedford, Mass.) andthe number of cells migrated expressed as fluorescent units.

In assays of inhibitory activity, cells in the upper wells of thechambers were suspended in varying concentrations (0.005, 0.05, 0.5,5.0, and 50 ng/ml) of MDC. The lower wells of the chamber were filledwith either medium alone or the chemotactic factors, MCP-1 or zymosanactivated serum (ZAS). Inhibition was assessed by comparing the numberof cells that migrated to MCP-1 or ZAS, in the absence of MDC, to thenumber of cells that migrated with increasing concentrations of MDC.Preparation of cells and quantitation of assays was performed exactly asdescribed above for the chemotaxis assays. The number of cells migratedwas expressed as fluorescent units.

As indicated in FIG. 2, MDC did not induce THP-1-derived mononuclearcell migration, but rather appeared to inhibit mononuclear cellmigration, at concentrations between 10 and 100 ng/ml. Other C-Cchemokines, such as MCP-1 and RANTES, typically induce maximal monocytechemotaxis within this concentration range.

As shown in FIG. 3, MDC, at concentrations of 0.001 to 100 ng/ml had nonet effect on granulocyte migration. In respect to this lack of effecton granulocyte chemotaxis, MDC is similar to other previously describedC-C chemokines.

The response of both macrophage and monocyte THP-1 cells to MDC is shownin FIG. 4. Macrophages (closed circles) migrated to MDC in a dosedependent manner, with optimal activity at 50 ng/ml. The decrease inmacrophage chemotactic response to MDC at higher concentrations (100ng/ml) reflects a desensitization of cells which is typical of mostchemotactic factors at high concentrations [Falk and Leonard, Infect.Immunol., 32:464468 (1981)]. Monocytic THP-1 cells (open circles)however, did not migrate to MDC.

The chemotactic activity of MDC for macrophages was further verified inexperiments utilizing elicited guinea pig peritoneal macrophages. MDCinduced a dose dependent migration of guinea pig macrophages (FIG. 5),at concentrations between 100 and 500 ng/ml. The concentrationsnecessary to induce the migration of guinea pig macrophages wasapproximately ten-fold of that for human cells (FIG. 4). Similardifferences in concentrations necessary for peak biological activity ofhuman chemokines in other species have been reported for MCP-1 byYashimura, J. Immunol., 150:5025-5032 (1993).

The results of these experiments suggest that the biological activitiesof MDC are linked to the differentiation of monocytes to macrophages. Incontrast to MCP-1 [Yoshimura, J. Immunol., 150:5025-5032 (1993)], MDCinduces macrophage but not monocyte chemotaxis.

The ability of MDC to attract macrophages indicates that this chemokinemight act to induce the focal accumulation of tissue macrophages. Theaccumulation of tissue macrophages in specific areas is important in theformation of granulomas, in which lung macrophages act to surround andenclose foreign particulates or relatively nondestructible bacterialpathogens such as Mycobacterium sp. [Adams, Am. J. Pathol., 84:164-191(1976)].

In certain conditions such as arthritis, the accumulation of macrophagesis understood to be detrimental and destructive. The ability of MDC topromote macrophage chemotaxis indicates a therapeutic utility for MDCinhibitors of the invention, to prevent, reduce, or eliminate macrophageaccumulation in tissues.

The results of the chemotaxis assays with human mononuclear cells,presented in FIG. 2, suggested that MDC might inhibit cell migration. Inthe absence of MDC, monocytic THP-1 cells migrate to MCP-1, as shown inFIG. 6 (MDC of 0 ng/ml). However, when cells are exposed to MDC, thechemotactic response to MCP-1 (closed circles) is decreased. MDC, atconcentrations of 0.005-0.5 ng/ml, inhibited monocyte chemotacticresponse to MCP-1. Although MDC inhibited the chemotactic response ofmonocytes to MCP-1, there was no significant effect of MDC onchemokinesis, or random migration, as reflected by the numbers of cellsmigrating to medium alone (open circles, RPMI with BSA), either in thepresence of absence of MDC.

The inhibitory activity of MDC on monocyte chemotaxis indicatestherapeutic utility for MDC in the treatment of several chronicinflammatory conditions (atherosclerosis, arthritis, pulmonary fibrosis)in which monocyte chemotaxis appears to play an important pathogenicrole. Enhancing the activity of MDC in such diseases might result in thedecreased migration of monocytes into tissues, thereby lessening theseverity of disease symptoms.

EXAMPLE 14 MDC In Vivo Tumor Growth Inhibition Assay

Tumor growth-inhibition properties of MDC are assayed, e.g., bymodifying the protocol described by Laning et al., J. Immunol.,153:4625-4635 (1994) for assaying the tumor growth-inhibitory propertiesof murine TCA3. An MDC-encoding cDNA is transfected by electroporationinto the myeloma-derived cell line J558 (American Type CultureCollection, Rockville, Md.). Transfectants are screened for MDCproduction by standard techniques such as ELISA (enzyme-linkedimmunoadsorbant assay) using a monoclonal antibody generated against MDCas detailed in Example 18. A bolus of 10 million cells from anMDC-producing clone is injected subcutaneously into the lower rightquadrant of BALB/c mice. For comparison, 10 million non-transfectedcells are injected into control mice. The rate and frequency of tumorformation in the two groups is compared to determine efficacy of MDC ininhibiting tumor growth. The nature of the cellular infiltratesubsequently associated with the tumor cells is identified by histologicmeans. In addition, recombinant MDC (20 ng) is mixed withnon-transfected J558 cells and injected (20 ng/day) into tumors derivedfrom such cells, to assay the effect of MDC administered exogenously totumor cells.

EXAMPLE 15 Intraperitoneal Injection Assay

The cells which respond to MDC in vivo are determined through injectionof 1-1000 ng of purified MDC into the intraperitoneal cavity of mice orother mammals (e.g., rabbits or guinea pigs), as described by Luo etal., J. Immunol., 153:46164624 (1994). Following injection, leukocytesare isolated from peripheral blood and from the peritoneal cavity andidentified by staning with the Diff Quick kit (Baxter, McGraw, Ill.).The profile of leukocytes is measured at various times to assess thekinetics of appearance of different cell types. In separate experiments,neutralizing antibodies directed against MDC (Example 18) are injectedalong with MDC to confirm that the infiltration of leukocytes is due tothe activity of MDC.

EXAMPLE 16 In vivo Activity Assay—Subcutaneous Injection

The chemoattractant properties of MDC are assayed in vivo by adaptingthe protocol described by Meurer et al., J. Exp. Med, 178:1913-1921(1993). Recombinant MDC (10-500 pmol/site) is injected intradermallyinto a suitable mammal, e.g., dogs or rabbits. At times of 4 to 24hours, cell infiltration at the site of injection is assessed byhistologic methods. The presence of MDC is confirmed byimmunocytochemistry using antibodies directed against MDC. The nature ofthe cellular infiltrate is identified by staining with Baxter's DiffQuick kit.

EXAMPLE 17 Melosuppression Activity Assays

The myelosuppressive activity of MDC is assayed by injection of MDC intomice or another mammal (e.g. rabbits, guinea pigs), e.g., as describedby Maze et al., J. Immunol., 149:1004-1009 (1992) for the measurement ofthe myelosuppressive action of MIP-1α. A single dose of 0.2 to 10 ug ofrecombinant MDC is intravenously injected into C3H/HeJ mice (JacksonLaboratories, Bar Harbor Me.). The myelosuppressive effect of thechemokine is determined by measuring the cycling rates of myeloidprogenitor cells in the femoral bone marrow and spleen The suppressionof growth and division of progenitor cells has clinical implications inthe treatment of patients receiving chemotherapy or radiation therapy.The myeloprotective effect of such chemokine treatment has beendemonstrated in pre-clinical models by Dunlop et al. Blood, 79:2221(1992).

An in vitro assay also is employed to measure the effect of MDC onmyelosuppression, in the same manner as described previously forderivatives of the chemokines interleukin-8 (IL-8) and platelet factor 4(PF-4). See Daly et al., J. Biol. Chem., 270:23282 (1995). Briefly, lowdensity (less than 1.077 g/cm) normal human bone marrow cells are platedin 0.3% agar culture medium with 10% fetal bovine serum (HyClone, Logan,Utah) with 100 units/ml recombinant human GM-CSF (R&D Systems,Minneapolis, Minn.) plus 50 ng/ml recombinant human Steel factor(Immunex Corp., Seattle, Wash.) in the absence (control) and presence ofMDC for assessment of granulocyte-macrophage precursors. For assessmentof granulocyte erythroid myeloid megakaryocyte colony forming units(CFU-GEMM) and erythroid burst forming units (BFU-E), cells are grown in0.9% methylcellulose culture medium in the presence of recombinant humanerythropoietin (1-2 units/ml) in combination with 50 ng/ml Steel factor.Plates are scored for colonies after incubation at 37° C. in lowered(5%) O₂ for 14 days. The combination of GM-CSF and Steel factor orerythropoietin and Steel factor allow detection of large colonies(usually >1000 cells/colony) which come from early, more immaturesubsets of granulocyte myeloid colony forming units (CFU-GM), CFU-GEMM,and BFU-E.

EXAMPLE 18 Antibodies to Human MDC

A. Monoclonal Antibodies

Recombinant MDC, produced by cleavage of a GST-MDC fusion protein asdescribed in Example 6, was used to immunize a mouse for generation ofmonoclonal antibodies. In addition, a separate mouse was immunized witha chemically synthesized peptide corresponding to the N-termunus of themature form of MDC (residues 1 to 12 of SEQ ID NO. 2). The peptide wassynthesized on an Applied Biosystem Model 473A Peptide Synthesizer(Foster City, Calif.), and conjugated to Keyhole Lympet Hemocyanine(Pierce), according to the manufacturer's recommendations. For theinitial injection to produce “Fusion 191” hybridomas, approximately 10μg of MDC protein or conjugated peptide was emulsified with Freund'sComplete Adjuvant and injected subcutaneously. At intervals of two tothree weeks, additional aliquots of MDC protein were emulsified withFreund's Incomplete Adjuvant and injected subcutaneously. Prior to thefinal prefusion boost, a sample of serum was taken from the immunizedmice. These sera were assayed by western blot to confirm theirreactivity with MDC protein. For a prefusion boost, the mouse wasinjected with MDC in PBS, and four days later the mouse was sacrificedand its spleen removed.

For the production of “Fusion 252” hybridomas, a mouse was immunizedwith the MDC(0-69) chemically synthesized peptide (See Example 11). OnDay 0, the mouse was pre-bled and injected subcutaneously at two siteswith 10 ug of MDC(0-69) in 200 ul complete Freund's adjuvant. On Day 22,the mouse was boosted with 30 ug of MDC(0-69) in 150 ul of incompleteFreund's adjuvant. On Day 40, the mouse was boosted with 20 ug MDC(0-69)in 100 ul of incomplete Freund's adjuvant. On day 54, blood was drawnand screened for anti-MDC antibodies via western blot, and reactivity,was observed against MDC. On days 127 through 130, the mouse wasinjected on each of four consecutive days with 10 ug of MDC(0-69) in avolume of 200 ul PBS. On day 131, the mouse was sacrificed and thespleen was removed for a fusion.

For the production of “Fusion 272” hybridomens, a mouse was treated in asimilar fashion as the mouse for fusion 252, except, on day 356, themouse was boosted with MDC(0-69) in incomplete Freund's adjuvant. Testbleeds were taken on day 367 and screened by ELISA On days 385, 386,387, and 388, the mouse was boosted with 5 μg injections of MDC(0-69).On day 389 the spleen was removed for a fusion.

The spleens were placed in 10 ml serum-free RPMI 1640, and single cellsuspensions were formed by grinding the spleens between the frosted endsof two glass microscope slides submerged in serum-free RPMI 1640,supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 100 units/mlpenicillin, and 100 μg/ml streptomycin (RPMI) (Gibco, Canada). The cellsuspensions were filtered through a sterile 70-mesh Nitex cell strainer(Becton Dickinson, Parsippany, N.J.), and were washed twice bycentrifuging at 200 g for 5 minutes and resuspending the pellet in 10 mlserum-free RPMI. Thymocytes taken from three naive Balb/c mice wereprepared in a similar manner and used as a Feeder Layer. NS-1 myelomacells, kept in log phase in RPMI with 100/c fetal bovine serum (FBS)cyclone Laboratories, Inc., Logan, Utah) for three days prior to fusion,were centrifuged at 200 g for 5 minutes, and the pellet was washed twiceas described above.

Spleen cells (2×10⁸) were combined with 4×10⁷ NS-1 cells andcentrifuged, and the supernatant was aspirated. The cell pellet wasdislodged by tapping the tube, and 2 ml of 37° C. PEG 1500 (50% in 75 mMHepes, pH 8.0) (Boehringer Mannheim) was added with stirring over thecourse of 1 minute, followed by the addition of 14 ml of serum-free RPMIover 7 minutes. An additional 16 ml RPMI was added and the cells werecentrifuged at 200 g for 10 minutes. After discarding the supernatant,the pellet was resuspended in 200 ml RPMI containing 15% FBS, 100 μMsodium hypoxanthine, 0.4 μM aminopterin, 16 μM thymidine (HAT) (Gibco),25 units/ml IL-6 (Boehringer Mannheim) and 1.5×10⁶ thymocytes/mil andplated into 10 Corning flat-bottom 96-well tissue culture plates(Corning, Corning N.Y.).

On days 2, 4, and 6, after the fusion, 100 μl of medium was removed fromthe wells of the fusion plates and replaced with fresh medium. On day 8,Fusion 191 was screened by ELISA, testing for the presence of mouse IgGbinding to MDC as follows. Immulon 4 plates (Dynatech, Cambridge, Mass.)were coated for 2 hours at 37° C. with 100 ng/well of MDC diluted in 25mM Tris, pH 7.5. The coating solution was aspirated and 200 ul/well ofblocking solution [0.5% fish skin gelatin (Sigma) diluted in CMF-PBS]was added and incubated for 30 min. at 37° C. The blocking solution wasaspirated and 50 μl culture supernatant was added. After incubation at37° C. for 30 minutes, and washing three times with PBS containing 0.05%Tween 20 (PBST), 50 μl of horseradish peroxidase conjugated goatanti-mouse IgG(fc) (Jackson ImmunoResearch, West Grove, Pa.) diluted1:7000 in PBST was added. Plates were incubated as above, washed fourtimes with PB ST, and 100 μL substrate, consisting of 1 mg/mlo-phenylene diamine (Sigma) and 0.1 μl/ml 30% H₂O₂ in 100 mM Citrate, pH4.5, was added. The color reaction was stopped after 5 minutes with theaddition of 50 μl of 15% H₂SO₄. A₄₉₀ was read on a plate reader(Dynatech). Fusions 252 and 272 were screened in a similar manner,except ELISA plates were coated with 50 ng/well of MDC.

Selected fusion wells were cloned twice by dilution into 96-well platesand visually scored for the number of colonies/well after 5 days. Themonoclonal antibodies produced by hybridomas were isotyped using theIsostrip system (Boehringer Mannheim, Indianapolis, Ind.).

Anti-MDC antibodies were characterized further by western blottingagainst recombinant MDC produced as described above in E. coli ormammalian CHO cells. To prepare the blot, approximately 3 μl ofsedimented cells (transformed E. coli producing MDC; transfected CHOcells producing MDC; untransformed E. coli (control); and untransfectedCHO cells (control)) were dissolved in standard sample preparationbuffer containing SDS (sodium dodecyl sulfate) and DTT (dithiolthreitol)(Sambrook et al.). After boiling, the lysates were fractionated viadenaturing SDS-PAGE (18% acrylamide, Tris Glycine gel, NOVEX) andelectroblotted to PVDF membranes (Millipore, Bedford, Mass.). MDCmonoclonal antibodies were diluted to 0.7 μg/ml in PBS for use in thewestern blotting, following standard techniques (Sambrook et al.). As anadditional control, the monoclonal antibodies were further tested forcross-reactivity on western blots of whole tissue lysates of human skin,tonsil, and thymus.

One anti-MDC monoclonal antibody, designated monoclonal antibody 191D,reacted strongly with recombinant MDC produced by both bacteria andmammalian cells. Further, this antibody displayed very little backgroundreactivity in preliminary screening against bacteria, the CHO mammaliancell line, or the whole human tissues tested. In addition, this antibodyshowed the ability to immunoprecipitate recombinant CHO-derived MDC,following standard immunoprecipitation protocols (Sambrook et al.).

Some background reactivity was observed in subsequent western analysesusing the anti-MDC monoclonal antibody 191D. Further anti-MDC monoclonalantibodies designated 252Y and 252Z (derived from Fusion 252), used at aconcentration of 4 ug/ml, showed less background and strong reactivitywith synthetic MDC at a concentration of 0.5 ng. No band was seen on thewestern blot with human tissue lysates of either colon, skin or tonsil,and background reactivity was minimal. The hybridomas that producemonoclonals 252Y and 252Z have been designated “hybridoma 252Y” and“hybridoma 252Z,” respectively.

Monoclonal antibody 272D, at 1 μg/ml, recognized 200 ng of wild type MDCby western blot, although less strongly than antibody 252Y. Antibody272D showed no background reactivity against lanes loaded with humanthymus whole cell lysate or human skeletal muscle lysate.

The hybridoma cell line which produces monoclonal antibody 191D(designated hybridoma 191D) has been deposited with the American TypeCulture Collection (ATCC), 10801 University Blvd., Manassas, Va.20110-2209 (USA) pursuant to the provisions of the Budapest Treaty (ATCCDeposit date: Jun. 4, 1996; ATCC Accession No. HB-12122). The hybridomacell lines that produce monoclonal antibodies 252Y and 252Z (designated“hybridoma 252Y” and “hybridoma 252Z”) were also deposited with the ATCCpursuant to the provisions of the Budapest Treaty (ATCC Deposit date:Nov. 19, 1997; ATCC Accession Nos. HB-12433 and HB-12434, respectively).The hybridoma cell line that produces monoclonal antibody 272D wasdeposited with the ATCC pursuant to the provisions of the BudapestTreaty on Mar. 27, 1998 (ATCC Accession No. HB-12498). Availability ofthe deposited materials is not to be construed as a license to practicethe invention in contravention of the rights granted under the authorityof any government in accordance with its patent laws.

B. Polyclonal Antibodies.

Polyclonal antibodies against MDC were raised in rabbits followingstandard protocols (Sambrook et al.). Recombinant MDC produced as a GSTfusion protein as described above was diluted in PBS, emulsified withFreund's Complete Adjuvant, and injected subcutaneously into rabbits. Atintervals of three and six weeks, additional MDC diluted in PBS wasemulsified with Freund's Incomplete Adjuvant and injected subcutaneouslyinto the same rabbits. Ten days after the third immunization, serum waswithdrawn from the rabbits and diluted ten-fold in Tris-buffered salinewith 0.5% Tween 20 (TBS-T, Sambrook et al.) for characterization viawestern blotting against recombinant MDC as described above.

In a similar set of experiments, polyclonal antisera was generated in arabbit against a 12-mer peptide corresponding to the amino-terminus ofmature MDC (SEQ ID NO: 2, positions 1-12). The resultant antiserum wascharacterized in Western blot experiments using synthetic MDC (matureform, residues 1-69); MDC(0-69); MDC(9-69); MDC-eyfy; and MDC-wvas (seeExample 11). The antiserum recognized all forms but the MDC(9-69)peptide.

C. MDC Detection Assay

Monoclonal antibodies 252Y and 252Z were employed in an MDC detectionassay as follows: Aliquots of the antibodies 252Y and 252Z werebiotinylated using NHS-LC-Biotin (Pierce) according to manufacturer'sinstructions. Immulon 4 ELISA plates were coated with one monoclonalantibody (252Y or 252Z, unbiotinylated) overnight at 4° C. The next day,the plates were blocked with 0.5% fish skin for 30 minutes at 37° C.Known quantities of MDC were loaded onto the plate for 30 minutes at 37°C. The plates were washed and coated with the other monoclonal antibody(biotinylated) for 30 minutes at 37° C. The plates were washed andloaded with streptavidin-HRP for 30 minutes at 37° C. The plates werethen developed and read on a Dynatech MR5000 plate reader. Preliminaryresults indicate that, by using the antibody pair 252Y and 252Z, MDC isdetectable in the concentration range of low nonograms to high picogramsper milliliter.

In a related set of experiments, an ELISA format was employed to examinethe relative affinity of antibodies 191D, 252Y, and 252Z for antigen.Antibodies were produced as ascites and purified over a protein A matrix(Prosep-A, Bioprocessing, LTD, Durham, England) according tomanufacturer's instructions. Eluted antibody was dialyzed against PBSand antibody concentration was assessed by A₂₈₀ measurements. MDC wascoated onto Immulon 4 plates in four-fold dilutions ranging from 2000 to0.4 ng/ml. After blocking and washing the plates as described above,each antibody was added at a constant concentration of 250 ng/ml, andA₂₈₀ measurements were taken to quantify antibody bound to the plates.The absorbance values for antibodies 252Y and 252Z were more thanfive-fold higher than those of antibody 191D (1.86 and 1.90 versus 0.34)at 2000 ng/ml MDC; more than seven-fold higher (1.22, 1.29, and 0.16,respectively) at 500 ng/ml, and more than three-fold higher (0.47, 0.47,and 0.13) at 125 ng/ml MDC. At 31 ng/rnl MDC, the A₂₈₀ measurements wereat background levels for all three antibodies.

D. Characterization of Epitopes Recognized by Antibodies 252Y and 252Zto recognize synthetic MDC

The ability of monoclonal antibodies 252Y and 252Z to recognizesynthetic MDC (mature form, residues 1-69) and MDC variants (MDC(0-69);MDC(9-69); MDC-eyfy; and MDC-wvas (see Example 11)) was analyzed viaWestern blot. One hundred to 500 nanograms of each synthetic peptide waselectrophoresed on a denaturing polyacrylamide gel, transferred, andprobed with antibody 252Y or antibody 252Z at a concentration of 1μg/ml. Immunoreactivity was visualized by incubating the probed blotwith horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G(Transduction Laboratories #M15345) at a concentration of 0.2 Fg/ml or1:5000 dilution in TRIS buffered saline with 0.1% Tween 20 (TBS Tw20)and 1% bovine serum albumin for 30 minutes at room temperature. The blotwas washed three times in the TRIS buffered saine/0.1% Tween 20 solutionand detection of antibody binding was measured by autoradiography (KodakHyperfilm) using electro-chemiluminescence (NEN Renaissance ECL # NEL102). Both monoclonal antibodies were observed to recognize wildtype MDCand the analogs MDC(0-69), MDC(9-69), and MDC-eyfy. However, antibody252Y and antibody 252Z both failed to recognize MDC-wvas, suggestingthat the epitope(s) recognized by these antibodies include(s) the wvmotif near the carbbxyl-terminus of MDC. This motif tends to be highlyconserved in all CC chemokines (see FIG. 1).

To further characterize the epitope(s) recognized by antibodies 252Y and252Z, an Immulon 4 plate was coated with MDC at 1.0 μg/ml. Afterblocking the plate with fish skin as described above in part C,unlabeled antibody 252Y, 252Z, or an isotype-matched control was addedat 5 μg/ml and incubated for 30 minutes at 37° C. Without washing,either biotinlyated antibody 252Y or 252Z was added at a concentrationof 0.25 μg/ml, and the plate was incubated an additional 30 minutes at37° C. Thereafter, the plate was washed and developed withstreptavidin-HRP. The results showed that either 252Y or 252Z wascapable of reducing the signal of either biotinlyated antibody ten-fold,as compared with the signal of either biotynilated antibody blocked withthe control antibody. These results further indicate that antibodies252Y and 252Z recognize similar or overlapping epitopes.

In contrast, unpurified supernatant from hydriboma 272D was tested in asimilar experiment for its ability to compete with biotynilated 252Y orbiotynilated 252Z, but was unable to reduce the signal of eitherantibody. Thus, monoclonal antibody 272D recognizes an epitope differentfrom that recognized by monoclonals 252Y and 252Z.

E. Antibodies 252Y and 252Z are Useful for Immunoprecipitating MDC

The following experiments were conducted which demonstrate a utility forantibodies 252Y and 252Z for immuprecipitation of MDC. Antibodies 252Y,252Z, and an irrelevant isotype-matched control were added separately ata concentration of 10 μg/ml to an extraction buffer (1% triton X-100, 10mM Tris base, 5 mM EDTA, 10 mM NaCl, 30 mM Na pyrophospate, 50 mM NaF,100 μM Na Orthovanadate, pH 7.6) containing 100 ng/ml MDC. These sampleswere incubated on ice for 1 hour. To precipitate the immune complexes,15 μl of protein G sepharose (Pharmacia Biotech # 17-0618-01) were addedto each sample and incubated on a rotation apparatus at 4° C. for 30minutes. The samples were then centrifuged to collect the protein Gsepharose/immune complexes, washed three times (1 ml each) in extractionbuffer, boiled/solubilized in 2×SDS-PAGE buffer, electrophoresed on an18% SDS-PAGE gel, and western blotted to PVDF membrane (Novex # LC2002).Nonspecific binding sites on the PVDF membrane were blocked with TBSTw20/1% BSA for 30 minutes at room temperature. The blot was then probedwith 1 μg/ml of antibody 252Y in TBS Tw20/1% BSA for 1 hour, washedthree times with TBS Tw20, probed with horseradish peroxidase-conjugatedgoat anti-mouse 1 gG in TBS Tw20/1% BSA for 30 minutes at roomtemperature, washed three times with TBS Tw20, and detected byautoradiography using ECL. Bands at approximately 8 kD were detected inthe 252Y and 252Z lanes but not in the negative isotype-matched controllane. Additionally, MDC was immunprecipitated from cell culturesupernatants containing RPMI (Rosell Park Memorial Institute—Gibco)medium with 10% fetal bovine serum spiked with 25 ng/ml MDC using thesame conditions stated above.

F. Humanization of Anti-MDC Monoclonal Antibodies

The activities of MDC as reported herein suggest numerous therapeuticindications for MDC inhibitors (antagonists). MDC-neutralizingantibodies (see Example 30) comprise one class of therapeutics useful asMDC antagonists. Following are protocols to improve the utility ofanti-MDC monoclonal antibodies as therapeutics in humans, by“humanizing” the monoclonal antibodies to improve their serum half-lifeand render them less immunogenic in human hosts (i.e., to prevent humanantibody response to non-human anti-MDC antibodies).

The principles of humanization have been described in the literature anda facilitated by the modular arrangement of antibody proteins. Tominimize the possibility binding complement, a humanized antibody of theIgG4 isotype is preferred.

For example, a level of humanization is achieved by generating chimericantibodies comprising the variable domains of non-human antibodyproteins of interest with the constant domains of human antibodymolecules. (See, e.g., Morrison and Oi, Adv. Immunol., 44:65-92 -(1989).The variable domains of MDC neutralizing anti-MDC antibodies are clonedfrom the genomic DNA of a B-cell hybridoma or from cDNA generated frommRNA isolated from the hybridoma of interest. The V region genefragments are linked to exons encoding human antibody constant domains,and the resultant construct is expressed in suitable mammalian hostcells (e.g., myeloma or CHO cells).

To achieve an even greater level of humanization, only those portions ofthe variable region gene fragments that encode antigen-bindingcomplementarity determining regions (“CDR”) of the non-human monoclonalantibody genes are cloned into human antibody sequences. [See, e.g.,Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature,332:323-327 (1988); Verhoeyen et al., Science, 239:1534-36 (1988); andTempest et al., Bio/Technology, 9:266-71(1991). If necessary, theβ-sheet framework of the human antibody surrounding the CDR3 regionsalso is modified to more closely mirror the three dimensional structureof the antigen-binding domain of the original monoclonal antibody. (SeeKettleborougfh et al., Protein Engin., 4:773-783 (1991); and Foote etal., J. Mol. Biol., 224:487-499 (1992).)

In an alternative approach, the surface of a non-human monoclonalantibody of interest is humanized by altering selected surface residuesof the non-human antibody, e.g., by site-directed mutagenesis, whileretaining all of the interior and contacting residues of the non-humanantibody. See Padlan, Molecular Immunol., 28(4/5):489-98 (1991).

The foregoing approaches are employed using MDC-neutralizing anti-MDCmonoclonal antibodies and the hybridomas that produce them, such asantibodies 252Y and 252Z, to generate humanized MDC-neutralizingantibodies useful as therapeutics to treat or palliate conditionswherein MDC expression is detrimental.

G. Human MDC-Neutralizing Antibodies from Phase Display

Human MDC-neutralizing antibodies are generated by phage displaytechniques such as those described in Aujame et al., Human Antibodies,8(4):155-168 (1997); Hoogenboom, TIBTECH, 15:62-70 (1997); and Rader etal., Curr. Opin. Biotechnol., 8:503-508 (1997), all of which areincorporated by reference. For example, antibody variable regions in theform of Fab fragments or linked single chain Fv fragments are fused tothe amino terminus of filamentous phage minor coat protein pIII.Expression of the fusion protein and incorporation thereof into themature phage coat results in phage particles that present an antibody ontheir surface and contain the genetic material encoding the antibody. Aphage library comprising such constructs is expressed in bacteria, andthe library is panned (screened) for MDC-specific phage-antibodies usinglabelled or immobilized SMC as antigen-probe.

H. Human MDC-Neutralizing Antibodies from Transgenic Mice

Human MDC-neutralizing antibodies are generated in transgenic miceessentially as described in Bruggemann and Neuberger, Immunol. Today,17(8):391-97 (1996) and Bruggemann and Taussig, Curr. Opin. Biotechnol.,8:455-58 (1997). Transgenic mice carrying human V-gene segments ingermline configuration and that express these transgenes in theirlymphoid tissue are immunized with an MDC composition using conventionalimmunization protocols. Hybridomas are generated using B cells from theimmunized mice using conventional protocols and screened to identifyhybridomas secreting anti-MDC human antibodies (e.g., as describedabove).

I. ELISA for Detecting and Monitoring Serum Concentrations of MDC

The measurement of endogenous levels of MDC is useful to monitor theimmune state of a patient, especially a patient who isimmunocompromized, in a hyperimmune state, or undergoing treatment withMDC neutralizing antibodies or other MDC antagonists.

A sensitive ELISA to measure MDC in biological fluids, for exampleserum, can be established using monoclonal antibodies, polyclonalantibodies, immuno-conjugates containing MDC ligands (for exampleheparin conjugates), or combinations thereof. For example, monoclonalantibodies 272D, 252Y and 252Z were employed in an MDC detection assayas described below.

Aliquots of the antibodies 252Y and 252Z were biotinylated usingNHS-LC-Biotin (Pierce) according to manufacturer's instructions. Immulon4 ELISA plates were coated with antibody 272D overnight at 4° C. Thenext day, the plates were blocked with 0.5% fish skin for 30 minutes at37° C. Known quantities of MDC(1-69) were loaded onto the plate for 30minutes at 37° C. The plates were washed and coated with either 252Y or252Z (biotinylated) for 30 minutes at 37° C. The plates were washed andloaded with streptavidin-HRP for 30 minutes at 37° C. The plates werethen developed and read on a Dynatech M000 plate reader. Preliminaryresults indicate that MDC is detectable in the concentration range oflow nanograms per milliliter in this ELISA format. It is expected thatuse of polyclonal antibodies for the capture antibody will lead to astill more sensitive ELISA assay.

EXAMPLE 19 Calcium Flux Assay

Changes in intracellular calcium concentrations, indicative of cellularactivation by chemokines, were monitored in several cell lines by anart-recognized calcium flux assay. Cells were incubated in 1 ml completemedia containing 1 μM Fura-2/AM (Molecular Probes, Eugene... Oreg.) for30 minutes at room temperature, washed once, and resuspended in D-PBS at˜1 cells/ml.

Two ml of suspended cells were placed in a continuously stirred cuvetteat 37° C. in a fluorimeter (AMINCO-Bowman Series 2, Rochester, N.Y.).The conceiitration of intracellular calcium was indicated byfluorescence, which was monitored at 510 nm emission wavelength whileswitching between excitation wavelengths of 340 nm and 380 nm every 0.5seconds. The ratio of the emissions from the 340 nm relative to the 380nm excitation wavelengths corresponds to the level of intracellularcalcium.

Cell lines measured by this assay included the following: the humanembryonic kidney cell line HEK-293 stably transfected with the putativechemokine receptor gene V28 [Raport et al., Gene, 163:295-299 (1995)];HEK-293 cells stably transfected with the chemokine receptor gene CCR5[Samson et al., Biochemistry, 35:3362-3367 (1996); see also co-owned,co-pending U.S. patent application Ser. No. 08/575,967, filed Dec. 20,1995, incorporated herein by reference, disclosing chemokine receptormaterials and methods, including CCR5 (identified therein as “88C”)],the human monocytic cell line THP-1, the human lung epithelial cell lineA-549; and the human fibroblast cell line IMR-90. None of these celllines fluxed calcium in response to the recombinant MDC protein. Aspositive controls, the HEK-293 transfectants responded strongly tothrombin, indicating that the assay was valid. In addition, the THP-1cells responded strongly to the commercially available chemokines MCP-1and MCP-3 (Peprotech, Rocky Hill N.J.) at a final concentration of 25ng/ml. No additional stimuli were tested on the A-549 or IMR-90 celllines.

EXAMPLE 20 Inhibition of HIV Proliferation

Several CC chemokines have been implicated in suppressing theproliferation of Human Immunodeficiency Virus (HIV), the causative agentof human Acquired Immune Deficiency Syndrome (AIDS). See Cocchi et al.,Science, 270:1811 (1995); Wikler et al., Science, 279:389-393 (1998).The HIV antiproliferative activity of MDC is measured by means such asthose described by Cocchi et al., in which a CD4⁺ T cell line is acutelyinfected with an HIV strain and cultured in the presence of variousconcentrations of MDC. After three days, a fresh dilution of MDC in theculture medium is added to the cells. At 5 to 7 days followinginfection, the level of HIV is measured by testing the culturesupernatants for the presence of HIV p24 antigen by a commercial ELISAtest (Coulter, Miami, Fla.).

One technical report teaches that MDC possesses an HIV antiproliferativeactivity. See Pal et al., Science, 278: 695-698 (1997). The agent usedin the study consisted of purified polypeptides that had been secretedfrom an immortalized cell line derived from CD8⁺ T cells from anHIV-1-infected individual. Pal et al. reported that the purified “nativeMDC” from this cell line possessed an NH₂-terminus corresponding to thetyrosine at position 3 of SEQ ID NO: 1. A “minor” sequence beginningwith the proline at position 2 of SEQ ID NO: 1 also was detected. Theauthors did not detect a peptide beginning with the glycine at position1 of SEQ ID NO: 1 in their “native MDC” composition. According to Pal etal., a reversed-phase HPLC fraction containing the “native MDC”suppressed the acute infection of CD8⁺ cell-depleted PBMCs byHIV-1_(IIIB) and various NSI HV isolates in a concentration-dependentfashion. Similar HIV suppressor activity was not observed insupernatants from other cell lines that appeared (from Northern blotstudies) to demonstrate equivalent MDC gene expression.

A. Use of MDC Antagonists to Inhibit HIV Proliferation

An acute HIV-1_(Bxl) infectivity assay reported in Pal et al. wasrepeated (100 TCID₅₀ units/well) using the macrophage cell line PM-1(1×10⁵ cells/well) and using purified mature MDC recombinantly expressedin CHO cells and having an amino acid sequence beginning at position 1of SEQ ID NO: 1 (see Example 10). Interestingly, mature MDC was found tohave no HIV suppressive activity. The same assay was performed withMDC(0-69) (See Example 11), an analog that exhibits properties of apartial MDC antagonist (see Example 19) in that it binds CCR4 withwild-type affinity, but exhibits substantially reduced capacity toinduce a calcium flux or induce chemotaxis. At a concentration of 1μg/ml, MDC(0-69) conferred a 58% and 67% reduction in the production ofinfectious particles (TCID₅₀ units measured on days 5 and 7). Thepositive control RANTES produced greater than 95% inhibition at 5 ng/ml.Without intending to be limited to a particular theory, one explanationfor these results is that mature MDC (1-69) induces HIV proliferation,and that the anti-proliferative effects of MDC(0-69) results from thisspecies competitively inhibiting the capacity of endogenous mature MDC(1-69) to stimulate HIV-1 production.

The effects of mature MDC and of MDC-neutralizing antibodies wereanalyze in Pal et al's acute BW-1_(BaL) (0.01 MOI/well) infectivityassay using peripheral blood mononuclear cells (PBMC, 1×10⁶ cells/well)depleted of CD8+ cells. The mature MDC (1-69) failed to inhibit p24production, as compared to a control murine IgG1 antibody. However, themurine monoclonal anti-MDC neutralizing antibodies 252Y (IgG1) and 252Zeach inhibited p24 production when tested separately at a concentrationof 2 μg/ml (37% and 28% inhibition, respectively). Again, oneexplanation for these data is that PBMC contain and produce endogenousMDC (1-69) that acts to stimulate HIV-1 functions, and that MDCantagonists inhibit this effect.

To confirm the apparent role of MDC as an HIV-1 agonist, an infectivityassay (such as that described in Pal et al.) is repeated using MDCneutralizing antibody and titrating exogenous mature MDC(1-69) into theassay wells. If native MDC(1-69) exerts an agonistic effect on HIV-1infectivity and/or proliferation, then it is expected that the antiviraleffect of the neutralizing antibody will be reduced with increasingamounts of mature MDC, and will be overwhelmed with the addition of amolar excess of MDC.

Collectively, these results provide a therapeutic indication for MDCantagonists for inhibiting proliferation of infectious retroviruses,especially HIV retroviruses. Such therapeutic methods and uses areintended as an aspect of the invention. For use in this context, theterm “MDC antagonist” includes any compound capable of inhibiting HIV-1proliferation in a manner analogous to MDC neutralizing antibodies, orMDC(O-69), or MDC(3-69). For example, anti-MDC antibodies (especiallyneutralizing antibodies, and preferably humanized antibodies) are highlypreferred MDC antagonists. Similarly, polypeptides that are capable ofbinding to MDC that comprise an antigen-binding fragment of an anti-MDCantibody are contemplated. Effective MDC analogs also are contemplatedas MDC antagonists. For example, N-terminal deletion analogs of MDC arecontemplated, especially deletion analogs having an amino acid sequenceconsisting of a portion of the amino acid sequence set forth in SEQ IDNO: 2 that is sufficient to bind to the chemokine receptor CCR4, theportion having an amino-terminus between residues 3 and 12 of SEQ ID NO:2. Likewise, analogs comprising a chemical addition to the aminoterminus to render said polypeptide antagonistic to MDC arecontemplated. The chemical addition may be added to the amino terminusof MDC(1-69) to form the analog, or to the amino terminus of an MDCanalog that has had amino acids deleted from its amino terminus (e.g.,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 residues deleted).

Additional classes of MDC antagonists useful in anti-HIV therapeuticmethods include antagonists derived from CCR4 or from other MDCreceptors. For example, a solubilized, MDC-binding version of CCR4 orCCR4 fragment is contemplated. Similarly, humanized antibodies thatblock but do not signal through CCR4 are contemplated as useful asanti-HIV therapeutics. Such antibodies are made using techniquesdescribed herein for making anti-MDC antibodies and/or techniques thathave been described in the art for generating antibodies to other seventransmembrane receptor proteins (e.g., using as an antigenCCR4-transfected cells that express CCR4 on their surface). See Wu etal., J. Exp. Med., 185:1681-1691 (1997).

Yet another class of MDC antagonists useful in anti-HIV therapeuticmethods of the invention include agents that have the effect oftransforming mature MDC(1-69) to antagonist forms in vivo, e.g., bymodifying the amino terminus of MDC. For example, administration of atherapeutically effective amount of the dipeptidyl aminopeptidase CD26is contemplated.

Therapeutically effective amounts of MDC antagonists (i.e., forinhibiting HIV infectivity and/or proliferation) are readily determinedusing standard dose-response studies. Moreover, determination of properdose and dosing is facilitated by anti-MDC antibodies of the invention(Example 18), which can be used in an ELISA or other standard assays tomonitor serum MDC levels in subjects receiving treatment. A therapeuticMDC neutralizing antibody should be administered in sufficient quantityand with sufficient frequency so as to maintain serum concentrations ofMDC below detectable levels. Doses of an MDC neutralizing antibody onthe order of 0.1 to 100 mg antibody per kilogram body weight, and morepreferably 1 to 10 mg/kg, are specifically contemplated. For humanizedantibodies, which typically exhibit a long circulating half-life, dosingat intervals ranging from daily to every other month, and morepreferably every week, or every other week, or every third week, arespecifically contemplated. Use of an IgG4 type humanizedMDC-neutralizing antibody is highly preferred, to minimize or eliminatethe possibility of inducing a complement reaction.

Moreover, determination of therapeutically effective MDC antagonists,doses, and dosing schedules is facilitated by dose-response studies inart-recognized in vivo models for HIV infection and proliferation, suchas studies in appropriate mice [Pettoello-Mantovani et al., Infect.Diseases, 177:337 (1998); J. M. McCune et al., “The Hematophtology ofHIV-1 Disease: Experimental Analysis in vivo,” in Human Hematopoiesis inSCID Mice, M. Roncarolo et al. (eds.), Landes Publishing Co., New York,N.Y., pp. 129-156 (1995); and McCune et al., “The SCID-hu mouse: a smallanimal model for HIV infection and antiviral testing,” in Progress inImmunol., VoL VII, Melchers et al. (eds.), Springer-VerlagBerlin-Heidelberg, pp. 1046-1049 (1989)) or primate models.

B. Use of TARC Antagonists to Inhibit HIV Proliferation

The foregoing experiments also suggest further analysis wherein an HIV-1infectivity assay is repeated using neutralizing antibodies directedagainst other beta chemokines. For those β-chemokines lacking anactivity towards TH² cells (analogous to MDC's activity toward suchcells), it is expected that chemokine-specific neutralizing antibodieswill behave much like the murine control IgG1 antibody above. However,for those β chemokines that possess an activity toward T_(H)2 cells thatis comparable to that of MDC (i.e., TARC), it is expected thatchemokine-specific neutralizing antibodies will behave much likeMDC-neutralizing antibodies and inhibit HIV-1 infectivity and/orproliferation. The use of TARC-neutralizing antibodies and/or other TARCinhibitors to suppress the infectivity and/or proliferation ofimmunodeficiency viruses is specifically contemplated as an aspect ofthe invention.

The nucleotide and deduced amino acid sequences of TARC have beenreported in the literature and are set forth herein in SEQ ID NOs: 42and 43. See Imai et al., J. Biol. Chem. 271: 21514-21521(1996); GENBANKACCESSION NO. D43767. TARC polypeptides and anti-TARC antibodies aresynthesized using procedures essentially as described herein for makingMDC and anti-MDC antibodies, or using procedures described in theliterature for TARC. [See Imai et al., J. Biol. Chem., 272: 15036-15042(1997); and Imai et al., J. Biol. Chem., 271: 21514-21521 (1996).] TheV-proliferative/anti-proliferative effects of TARC polypeptides (e.g.,native mature TARC and TARC analogs, especially amino-terminal deletionand addition analogs) and TARC-neutralizing antibodies are assayedessentially as described in Pal et al. or Cocchi et al.

Based on the theory that the HIV antiproliferative efficacy of MDCantagonists is mediated by blocking the signaling of MDC through CCR4 intarget cells that express CCR4, it is further contemplated thatantibodies to any other chemokine that known or is discovered to signalthrough CCR4 will be useful as anti-HIV therapeutics of the invention.

EXAMPLE 21 Effects of MDC on Fibroblast Proliferation

In addition to their ability to attract and activate leukocytes, somechemokines, such as IL-8, have been shown to be capable of affecting theproliferation of non-leukocytic cells [see Tuschil, J. Invest.Dermatol., 99:294-298 (1992)]. Fibroblasts throughout the body areimportant to the structural integrity of most tissues. The proliferationof fibroblasts is essential to wound healing and response to injury butcan be deleterious as well, as in the case of chronic inflammatorydiseases, such as pulmonary fibrosis [Phan, in: Immunology ofInflammation, Elsevier (1983), pp. 121-162].

In vitro cell proliferation assays were utilized to assess the effectsof MDC on the proliferation of fibroblasts. Human fibroblasts (CRL-1635)were obtained from ATCC and maintained in culture in DMEM with 10% FBSand 1% antibiotics. Proliferation assays were performed and quantitatedas previously described in the art by Denholm and Phan, Amer. J Pathol.,134:355-363 (1989). Briefly, on day 1, 2.5×10³ cells/well were platedinto 96 well plates in DMEM with 10% FBS. Day 2: twenty-four hours afterplating, medium on cells was changed to serum-free DMEM. Day 3: mediumwas removed from cells and replaced with MDC diluted in DMEM containing0.4% FBS. Day 5: one microCurie of ³H-thymidine was added per well andincubation continued for an additional 5 hours. Cells were harvestedonto glass fiber filters. Cell proliferation was expressed as cpm of³H-thymidine incorporated into fibroblasts. Controls for this assayincluded the basal medium for this assay, DMEM with 0.4% FBS as thenegative control, and DMEM with 10% FBS as the positive control.

As shown in FIG. 7, MDC treatment decreased the proliferation offibroblasts in a dose dependent manner. Similar inhibition of fibroblastproliferation was observed with both MDC purified from CHO cells (closedcircles) and chemically synthesized MDC (open circles). Thefibroblast-antiproliferative effect of MDC indicates a therapeuticutility for MDC in the treatment of diseases such as pulmonary fibrosisand tumors, in which enhanced or uncontrolled cell proliferation is amajor feature.

EXAMPLE 22 Cell Proliferation Assays

The effects of MDC upon the proliferation of epithelial cells, T cells,fibroblasts, endothelial cells, macrophages, and tumor cells are assayedby methods known in the art, such as those described in Denholn et al.,Amer. J Pathol., 134:355-363 (1989), and “In Vitro Assays of LymphocyteFunctions,” in: Current Protocols Immunology, Sections 3-4, Wiley andSons (1992), for the assay of growth factor activities. In thesemethods, enhancement or inhibition of cell growth and the release ofgrowth factors are measured.

MDC effects on the proliferation of epithelial cells and endothelialcells are assayed using the same procedures as those described above forfibroblasts (Example 21).

The effects on the proliferation of T cells are determined usingperipheral blood lymphocytes. Mononuclear cells are isolated fromperipheral blood as described in Denholm et al., Amer. J. Pathol,135:571-580 (1989); cells are resuspended in RPMI with 10% FBS andincubated overnight in plastic tissue culture flasks. Lymphocytes remainin suspension in these cultures and are obtained by centrifugation ofculture medium. One hundred thousand lymphocytes are plated into eachwell of a 96 well plate and incubated for three days in medium (RPMIplus 10% FBS) containing 1 μg/ml PHA with or without 50, 125, 250 or 500ng/ml of MDC. One microCurie of ³H thymidine is added during the last 18hours of incubation. Cells are harvested and proliferations expressed asdescribed for fibroblasts in Example 21.

The effects of MDC on macrophage proliferation are determined usingelicited guinea pig peritoneal macrophages, obtained as described abovein Example 13. Macrophages are plated into 96 well plates at a densityof one hundred thousand cells per well in RPMI with 10% FBS, andincubated 2 hours to allow cells to adhere. Medium is then removed andreplaced with fresh medium with or without 50, 125, 250 or 500 ng/ml ofMDC. Cells with MDC are incubated three days, and proliferation isdetermined as described above for lymphocytes.

Chemokine-mediated control of the proliferation of these cell types hastherapeutic implications in enhancing tissue repair following injury,and in limiting the proliferation of these cells in chronic inflammatoryreactions such as psoriasis, fibrosis, and atherosclerosis, and inneoplastic conditions.

EXAMPLE 23 In Vivo Fibroblast Proliferation Assay

The anti-proliferative effects of MDC upon fibroblasts are determined invivo by the methods known in the art, such as those reported by Phan andFantone, Amer. J. Pathol., 50:587-591 (1984), which utilize a rat modelof pulmonary fibrosis in which the disease is induced by bleomycin. Thismodel is well-characterized and allows for the assessment of fibroblastproliferation and collagen synthesis during all stages of this disease.

Briefly, rats are divided into four treatment groups: 1) controls, givenintratracheal injections of normal saline; 2) saline-injected rats whichalso receive a daily intraperitoneal injection of 500 ng of MDC insaline; 3) bleomycin-treated, given an intratracheal injection of 1.5mg/kg bleomycin (Calbiochem, Palo Alto, Calif.); and 4)bleomycin-treated rats which also are given a daily intraperitonealinjection of 500 ng of MDC.

Three rats per group are sacrificed at 4, 7, 14, 21, and 28 days afterthe initial intratracheal injections. Lungs are removed and samples ofeach lobe taken for histological examination and assays of collagencontent.

EXAMPLE 24 MDC Chromosomal Localization

A 20 kb genomic fragment containing the human MDC gene was labelled withdigoxigenin by nick translation and used as a probe for fluorescence insitu hybridization of human chromosomes (Genome Systems, Inc., St.Louis, Mo.). The labelled probe was hybridized to normal metaphasechromosomes derived from PHA-stimulated peripheral blood lymphocytes.Reactions were carried out in the presence of sheared human DNA in 50%formamide, 10% dextran sulfate, 30 mM sodium chloride, 3 mM sodiumcitrate, and 0.1% sodium dodecyl sulphate. Hybridization signals weredetected by treating slides with fluoresceinated anti-digoxigeninantibodies, followed by counter-staining with4,6-diamidino-2-phenylindole. An initial hybridization experimentlocalized the gene to the q terminus of a group E chromosome.

A genomic probe that specifically hybridizes to the short arm ofchromosome 16 was used to demonstrate co-hybridization of chromosome 16with the MDC probe. A total of 80 metaphase cells were analyzed with 61exhibiting specific labeling. The MDC probe hybridized to a regionimmediately adjacent to the heterochromatic/euchromatic boundary,corresponding to band 16q13. The gene encoding TARC also is localized inthis region. See Normiyama et al., Genomics, 40: 211-213 (1997).

These chromosomal mapping data indicate a utility of MDC-encodingpolynucleotides as a chromosomal marker. Contiguous fragments of SEQ IDNO: 1 of at least 15 nucleotides, and more preferably at least 20, 25,50, 75, 100, 150, 200, 500, or more nucleotides, and the complements ofsuch fragments, are specifically contemplated as probes of theinvention. Moreover, probes having partial degeneracy from SEQ ID NO: 1are contemplated as being useful as well. Probes having preferably atleast 90%, and more preferably 95%, 96% 97%, 98%, 99%, or moresimilarity to SEQ ID NO: 1 are preferred as probes of the invention.

EXAMPLE 25 MDC is a High-Affinity Ligand for CCR4

The chemokine receptor designated CCR4 has been characterized previously[Power et al., J. Biol. Chem., 270: 19495-19500 (1995)], and shown tobind the CC chemokine TARC (Thymus and Activation-Regulated Chemokine,Genbank Accession No. D43767). See Imai et. al., J. Biol. Chem., 272:15036-15042 (1997); and Imai et al., J. Biol. Chem., 271: 21514-21521(1996). The cDNA and deduced amino acid sequences of human CCR4 are setforth in SEQ ID NOs: 33 and 34, and are deposited with Genbank(Accession No. X85740). The following experiments were performed thatdemonstrate that MDC is a high affinity ligand for CCR4.

A. Preparation of CCR4-Transfected Cells

The murine pre-B cell line L1.2 [See, e.g., Gallatin et al., Nature,304:30-34 (1983)] maintained in RPMI 1640 media supplemented with 10%fetal calf serum; was selected for transformation with the CCR4expression vector described in Imai et al., J. Biol. Chem., 272:15036-15042 (1997), incorporated herein by reference. L1.2 cells werestably transfected as described previously by electroporation with 10 μglinearized plasmid at 260 V, 960 microfarads using a Gene Pulser(BioRad). See Imai et al., J. Biol. Chem., 272: 15036-15042 (1997). Itwill be apparent that other cell lines in the art are suitable for CCR4transfection for the following assays. For example, 293 cell lines havebeen transfected with CCR4 cDNA and employed effectively in calcium Fluxassays.

B. Preparation of Recombinant Chemokines

The mature sequences of both MDC and TARC were chemically synthesized byGryphon Sciences (South San Francisco Calif.) using t-butyl-oxycarbonylchemistries on a peptide synthesizer (430A; Applied Biosystems).Lyophilized protein was dissolved at 10 mg/ml in 4 mM HCl andimmediately diluted to 0.1 mg/ml in phosphate-buffered saline plus 0.1%bovine serum albumin (BSA) for storage at −80° C.

Recombinant MDC also was expressed as a fusion protein with the secretedform of placental alkaline phosphatase (SEAP) in the expression vectorpcDNA3 (Clontech, Palo Alto Calif.). A similar TARC-SEAP fusion proteinis described in Imai et al. (1997). Briefly, the coding region of MDC,followed by a sequence encoding a five amino acid linker(Ser-Arg-Ser-Ser-Gly) was fused in-frame to a sequence encoding matureSEAP, followed by a sequence encoding a (His)₆ tag. The MDC-SEAPexpression plasmid was transfected into COS cells by the DEAE Dextranmethod. See Sambrook et al., Molecular Cloning. A Laboratory Manual,Cold Spring Harbor Laboratory, Cold Spring Harbor; NY (1989). Thetransfected cells were cultured in Dulbecco's modified Eagle's mediumsupplemented with 10% fetal calf serum. Twenty-four hours aftertransfection, the serum levels were reduced from 10% to 1%. After 3-4days, the culture supernatants were collected, centrifuged, filteredthrough a 0.45 micron membrane, and stored at 4° C. The concentration ofMDC-SEAP in the filtered supernatant was determined by comparison withthe reported specific activity of secreted placental alkalinephosphatase [Berger et al., Gene, 66: 1-10 (1988)], and confirmed usingknown concentrations of TARC-SEAP [Imai et al., (1997)) as an internalreference standard.

C. CCR4 Binding Assays

The MDC-SEAP was used as a probe to examine MDC binding toCCR4-transfected L1.2 cells. For displacement and saturationexperiments, transfected L1.2 cells (approx. 3×10⁵) were incubated forone hour at 16° C. in the presence of 0.5 nM MDC-SEAP in the presence orabsence of various concentrations of unlabeled chemokines in 200 μlbinding buffer (RPMI 1640 media containing 25 mM HEPES, pH 7.4, 1% BSA,and 0.02% sodium azide). Following incubation, the cells were washedfour times in binding buffer and lysed in 50 μl of 10 mM Tris-HCl, pH8.0, and 1% Triton X-100. Samples were heated at 65° C. for 15 minutesto inactivate cellular phosphatases, centrifuged, and stored at −20° C.until assayed.

Alkaline phosphatase activity in 10 μl of sample was determined by achemiluminescence assay using the Great Escape Detection kit (Clontech,Palo Alto, Calif.) according to the manufacturer's instructions. Thesaturation binding curve was fitted (Table Curve™) using the Hillequation y=a(x^(c))/(x^(c)+b^(c)), where y is the amount of ligandbound, α is the maximum amount of ligand bound, x is the concentrationof ligand, b is the concentration of ligand at which 50% of receptorsites are occupied (K_(D)), and c is the Hill coefficient. Bindingcompetition curves were fitted (TableCurve™) using a three-parameterlogistic model described by the equation y=a/[1]+(x/b)^(c)], where y isthe amount of labelled ligand bound, α is the maximum amount of labelledligand bound, x is the concentration of the competitive chemokine, b isthe IC₅₀, and c is a parameter that determines the slope of the curve atthe IC₅₀.

These binding assays demonstrated that MDC-SEAP bound to CCR4-expressingcells. This binding was to a single high affinity site with a K_(d) of0.18 nM, as demonstrated by Scatchard analysis. Binding of MDC-SEAP wascompetitively inhibited with increasing concentrations of unlabeled MDCor TARC. The IC₅₀ for MDC was 0.65 nM, while the IC₅₀ for TARC was 2.1nM. These data suggest that both MDC and TARC recognize a common bindingsite on CCR4, and that MDC has more than three-fold higher affinity thanTARC for CCR4.

To examine the specificity of MDC binding to CCR4, six additionalchemokines (MCP-1, MCP-3, MCP4, RANTES, MIP-1α, and MIP-1β) were testedfor competition of MDC-SEAP binding. A 200-fold molar excess of eachchemokine was tested for competition with a constant quantity ofMDC-SEAP (0.5 nM) The additional chemokines did not compete for bindingof MDC SEAP to CCR4. In contrast, unlabeled MDC and TARC both blockedbinding of MDC-SEAP to CCR4 transfectants.

D. Calcium Mobilization Assay

Imai et al. (1997) showed that TARC signals through CCR4 by inducingcalcium mobilization. To determine the ability of MDC to cause signalingthrough chemokine receptors, we examined calcium mobilization in L1.2cells recombinantly expressing CCR1, CCR2B, CCR3, CCR4, CCR5, CCR6, orCCR7.

Transfected L1.2 cells were suspended at a concentration of 3×10⁶cells/ml in Hank's balanced salt solution supplemented with 1 mg/m BSAand 10 mM HEPES, pH 7.4. Cells were incubated with 1 μM fura-PE3-AM(Texas Fluorescence Labs) at room temperature for 1 hour in the dark.After washing twice, cells were resuspended at a concentration of2.5×10⁶ cells/ml. To measure intracellular calcium, 2 ml of cells wereplaced in a quartz cuvette in a Perkin-Elmer LS 50B spectrofluorimeter.Fluorescence was monitored at 340 nm (excitation wavelength 1), 380 nm(excitation wavelength 2), and 510 nm (emission wavelength) every 200ms.

In these experiments, MDC did not cause calcium flux in L1.2 cellstransfected with CCR1, CCR2B, CCR3, CCR5, CCR6, or CCR7, whereas each ofthese transfected cell lines responded to its known cognate ligand. Incontrast, L1.2 cells transfected with CCR4 produced a strong calciumflux when stimulated with 10 nM MDC. Similar to other G protein-coupledreceptors, CCR4 was refractory to subsequent stimulation with the sameconcentration of MDC. Ten nanomolar MDC also completely desensitizedCCR4 transfectants to subsequent 10 nM TARC treatment. However,pre-treatment of CCR4-transfected L1.2 cells with TARC did notdesensitize the receptor to subsequent stimulation with MDC. The signalproduced by initial TARC stimulation was of lower intensity than boththe primary MDC signal and the MDC signal secondary to TARC stimulation.These results further confirm that MDC is a ligand for CCR4.

E. Chemotaxis Assay

We next examined the ability of MDC and TARC to induce migration ofCCR4-transfected L1.2 cells. Approximately 10⁶ CCR4-transfected L1.2cells, resuspended in 0.1 ml RPMI 1640 media with 0.5% BSA, were loadedin the upper wells of a transwell chamber (3 μm pore size, Costar).Untransfected L1.2 cells were used as a control. Test chemokines wereadded to the lower wells at a concentration of 0-100 nM in a volume of0.6 ml. After 4 hours at 37° C., cells in the lower chamber werecollected and counted by FACS.

Both MDC and TARC induced migration of CCR4-transfected L1.2 cells. Bothchemokines produced classic bell-shaped migration responses with maximalmigration at about 10 nM. The migration observed with MDC wassignificantly higher than that for TARC, with MDC inducing migration ofgreater than 7% of input cells versus less than 3% migration for TARC.Untransfected L1.2 cells failed to migrate when treated with MDC. Thesechemotaxis results further confirm that both MDC and TARC are functionalligands for CCR4.

F. Conclusion

Collectively, the foregoing experiments provide compelling evidence thatMDC acts as a high affinity ligand for the chemokine receptor CCR4.

As described below in Example 32, CCR4 has been found to be abundantlyand nearly exclusively expressed on antigen-specific T_(H)2 helper Tcells. Such cells are particularly susceptible to HIV-1 infection. (SeeMaggi et al., Science, 265:244-252 (1994).) The identification herein ofa high affinity MDC receptor on HIV-susceptible T cells indicates aputative mechanism/pathway through which MDC(1-69) exerts its agonisticactivity relating to enhanced HIV-1 infectivity and or viral productionin infected cells (see Example 20), and likewise indicates a target fortherapeutic intervention. Without intending to be limited to aparticular theory, MDC-mediated activation of T_(H)2 cells, through theCCR4 receptor, is postulated to enhance infectivity and/or production ofHIV-1 virus, in a manner analogous to the increased infectivity that haspreviously been observed for activated target cells. See Woods et al.,Blood, 89: 1635-1641 (1997); and Roederer et al., J. Clin. Invest.,99(7): 1555-1564 (1997).

EXAMPLE 26 MDC Modulator Assays

Modulators of MDC activity may be useful for the treatment of diseasesor symptoms of diseases wherein MDC plays a role. Such modulators may beeither agonists or antagonists of MDC binding. The following receptorbinding assays provide procedures for identifying such MDC modulators.

MDC is labelled with a detectable label such as ¹²⁵I, ³H, ¹⁴C, biotin,or Europium. A preparation of cell membranes containing MDC receptors isprepared from natural cells that respond to MDC, such as humanmacrophages, phorbol ester-stimulated THP-1 cells, human fibroblasts,human fibroblast cell lines, or guinea pig macrophages. (Alternatively,a recombinant receptor preparation is made from cells transfected withan MDC receptor cDNA, such as a mammalian cell line transfected with acDNA encoding CCR4 and expressing CCR4 on its surface.) The membranepreparation is exposed to ¹²⁵I-labelled MDC, for example, and incubatedunder suitable conditions (e.g., ten minutes at 37° C.). The membranes,with any bound ¹²⁵I-MDC, are then collected on a filter by vacuumfiltration and washed to remove unbound ¹²⁵I-MDC. The radioactivityassociated with the bound MDC is then quantitated by subjecting thefilters to liquid scintillation spectrophotometry.

The specificity of MDC binding may be confirmed by repeating theforegoing assay in the presence of increasing quantities of unlabeledMDC, and measuring the level of competition for binding to the receptor.These binding assays also can be employed to identify modulators of MDCreceptor binding.

The foregoing receptor binding assay also may be performed with thefollowing modification: in addition to labelled MDC, a potential MDCmodulator is exposed to the membrane preparation. In this assayvariation, an increased level (quantity) of membrane-associated labelindicates the potential modulator is an activator of MDC binding; adecreased level (quantity) of membrane-associated label indicates thepotential modulator is an inhibitor of MDC receptor binding. This assaycan be utilized to identify specific activators and inhibitors of MDCbinding from large libraries of chemical compounds or natural products.Rapid screening of multiple modulator candidate compounds simultaneouslyis specifically contemplated.

EXAMPLE 27 Assay to Identify Modulators of the MDC/CCR4 Interaction

The discovery that CCR4 acts as an MDC receptor prompted the developmentof the following additional assays to identify modulators of theinteraction between MDC and CCR4. Such assays are intended as aspects ofthe present invention.

A. Direct Assay

In one embodiment, the invention comprehends a direct assay formodulation (potentiation or inhibition) of MDC-receptor binding. In onedirect assay, membrane preparations presenting the chemokine receptorCCR4 in a functional conformation are exposed to either MDC alone or MDCin combination with potential modulators.

For suitable membrane preparations, tissue culture cells, such as 293 orK-562 cells (ATCC CRL-1573 and CCL-243, respectively), are transfectedwith an expression vehicle encoding the MDC receptor CCR4. Cells thatexpress the receptor are selected and cultured, and a membranepreparation is made from the transfected cels expressing the chemokinereceptor. By way of example, suitable membrane preparations are made byhomogenizing cells in TEM buffer (25 mM Tris-HCL, pH 7.4, 1 mM EDTA, 6mM MgCl₂, 10 μM PMSF, 1 μg/ml leupeptin) The homogenate is centrifugedat 800×g for 10 minutes. The resulting pellet is homogenized again inTEM and re-pelleted. The combined supernatants are then centrifuged at100,000×g for one hour. The pellets containing the membrane preparationsare resuspended in TEM at 1.5 mg/ml.

Membrane preparations are exposed to labelled MDC (e.g., MDC labelledwith I¹²⁵ or other isotope, MDC prepared as an MDC-secreted alkalinephosphatase fusion protein, or MDC labelled in some other manner) eitherin the presence (experimental) or absence (control) of one or morecompounds to be tested for the ability to modulate MDC-receptor bindingactivity. To practice the assay in standard 96-well plates, an exemplaryreaction would include 2 μg of the membrane preparation, 0.06 nM ofradio-labelled MDC, and 0.01 to 100 μM of one or more test compounds, ina reaction buffer comprising 50 mM HEPES, pH 7.4, 1 mM CaCl₂, 5 mMMgCl₂, and 0.1% BSA. The reactions are then incubated under suitableconditions (e.g., for 1-120 minutes, or more preferably 10-60 minutes,at a temperature from about room temperature to about 37° C.).

After incubation, the membranes, with any bound MDC and test compounds,are collected on a filter by vacuum filtration and washed to remove anyunbound ligand and test compound. Thereafter, the amount of labelled MDCassociated with the washed membrane preparation is quantified. In anembodiment wherein the label is a radioisotope, then bound MDCpreferably is quantified by subjecting the filters to liquidscintillation spectrophotometry. In an embodiment wherein anMDC-alkaline phosphatase fusion protein is employed, alkalinephosphatase activity is measured using, for example, the “Great Escape”detection kit (Clontech, Palo Alto, Calif.) according to themanufacturer's instructions. The amount of label (e.g., scintillationcounts or alkaline phosphatase activity) associated with the membranesis proportional to the amount of labelled MDC bound thereto. If thequantity of bound, labelled MDC observed in an experimental reaction isgreater than the amount observed in the corresponding control, then theexperimental reaction is scored as containing one or more putativeagonists (i.e., activators, potentiators) of MDC receptor binding. Ifthe quantity of bound, labelled MDC observed in an experimental reactionis less than the amount observed in the corresponding control, then theexperimental reaction is scored as containing one or more putativeantagonists (inhibitors) of MDC receptor binding.

The specificity of modulator binding may be confirmed by repeating theforegoing assay in the presence of increasing quantities of unlabeledtest compound and noting the level of competition for binding to thereceptor. The assay may also be repeated using labelled modulatorcompounds, to determine whether the modulator compound operates bybinding with the MDC receptor.

B. Indirect GDP Assay

In another embodiment, the invention comprehends indirect assays foridentifying modulations of MDC receptor binding that exploit thecoupling of chemokine receptors to G proteins. As reviewed in Linder etal., Sci. Am., 267: 56-65 (1992), during signal transduction, anactivated receptor interacts with and activates a G protein. The Gprotein is activated by exchanging GDP for GTP. Subsequent hydrolysis ofthe G protein-bound GTP deactivates the G protein. Therefore, one canindirectly assay for G protein activity by monitoring the release of³²P_(i) from [γ-³²P]-GTP.

For example, approximately 5×10⁷ HEK-293 cells that have beentransformed or transfected (e.g., with a CCR4 expression vector) toexpress CCR4 are grown in MEM+10% fetal calf serum (FCS). The growthmedium is supplemented with 5 mCi/ml [³²P]-sodium phosphate for 2 hoursto uniformly label nucleotide pools. The cells are subsequently washedin a low-phosphate isotonic buffer.

An experimental aliquot of washed cells is exposed to MDC in thepresence of one or more test compounds, while a control aliquot of cellsis exposed to MDC, but without exposure to the test compound. Followingan incubation period (e.g., 10 minutes, 37° C.), cells are pelleted andlysed, and nucleotide compounds are fractionated using, e.g., thin layerchromatography (TLC) developed with 1 M LiCl. Labelled GTP and GDP areidentified in the TLC by developing known GTP and GDP standards inparallel. The labelled GTP and GDP are then quantified byautoradiographic techniques that are standard in the art.

In this assay, the extent of MDC interaction with its receptor isproportional to the levels of ³²P-labelled GDP that are observed,thereby permitting the identification of modulators of MDC-CCR4 binding.An intensified signal resulting from a relative increase in GTPhydrolysis producing ³²P-labelled GDP, indicates a relative increase inreceptor activity. The intensified signal therefore identifies thepotential modulator as an activator of MDC-CCR4 activity, or possibly asan MDC mimetic. Conversely, a diminished relative signal for³²P-labelled GDP, indicative of decreased receptor activity, identifiesthe potential modulator as an inhibitor of MDC receptor binding or aninhibitor of MDC-induced CCR4 signal transduction.

C. cAMP Assay

The activities of G protein effector molecules (e.g., adenylyl cyclase,phospholipasE C, ion channels, and phosphodiesterases) are also amenableto assay. Assays for the activities of these effector molecules havebeen previously described. For example, adenylyl cyclase, whichcatalyzes the synthesis of cyclic adenosine monophosphate (cAMP), isactivated by G proteins. Therefore, MDC binding and activation of CCR4that activates a G protein, which in turn. activates adenylyl cyclase,can be detected by monitoring cAMP levels in a host cell thatrecombinantly expresses CCR4.

Host cells that recombinantly express CCR4 are preferred for use in theassay. The host cells are incubated in the presence of either MDC aloneor MDC plus one or more test compounds as described above. The cells arelysed, and the concentration of cAMP is measured by a suitable assay,such as a commercial enzyme immunoassay. For example, the BioTrak Kit(Amersham, Inc., Arlington Heights, Ill.) provides reagents for asuitable competitive immunoassay for cAMP.

An elevated level of intracellular cAMP in a test reaction relative to acontrol reaction is attributed to the presence of one or more testcompounds that increase or mimic MDC-induced CCR4 activity, therebyidentifying a potential activator compound. A relative reduction in theconcentration of cAMP would indirectly identify an inhibitor ofMDC-induced CCR4 activity.

It will be apparent to those in the art that the foregoing assays may beperformed using MDC analogs described herein. Moreover, variations ofthe foregoing assays will be apparent to those in the art. Anyvariations that utilize both MDC and CCR4, and especially thosevariations which utilize MDC and cells that recombinantly express CCR4,are intended as aspects of the invention.

While the use of human MDC and CCR4 comprises a highly preferredembodiment, it will be apparent that the source organism for MDC andCCR4 is not a limiting factor, and the foregoing assays may be practicedeffectively with MDC and/or with CCR4 that are derived from non-humanorganisms. By way of example, rat and mouse MDC are taught herein; and aMus musculus chemokine receptor 4 sequence has been reported in the art.See Hoogewerf et al., Biochem. Biophys. Res. Comm., 218(1): 337-343, andGenBanK Accession No. X90862. Moreover, the methods used herein toobtain rat and mouse MDC are employable to obtain MDC or CCR4 from otherorganisms.

Moreover, evidence exists that there is at least one additional receptorthat recognizes MDC. For example, MDC stimulates migration of dendriticcells and IL-2 activated natural killer cells. Godiska et al., J. Exp.Med., 185: 1595-1604 (1997), incorporated herein by reference. Thismigration is not likely to be mediated by CCR4, since CCR4 appears to beexpressed primarily on T cells, but not on monocytes or NK cells. SeeImai et al. (1997). Consistent with this, CCR4 clones were representedvery rarely in a human macrophage cDNA library (less than one in amillion clones). Variations of the assays reported herein that utilizeMDC with other MDC receptors also are intended as aspects of theinvention.

Additionally, it will be apparent that the protocols described inpreceding examples for assaying MDC biological activities (in-vivo orwith respect to specific cell types in vitro) are useful as assays forMDC modulators. In a highly preferred embodiment, a compound is firstidentified as a candidate MDC modulator using any of the assaysdescribed in Examples 26 and 27. Compounds that modulate MDC-receptoractivity in one or more of these initial assays are further screened inany of the protocols described in preceding examples, to determine theability of the compounds to modulate the MDC biological activities towhich those examples specifically relate.

EXAMPLE 28 Non-Human Vertebrate MDC cDNAs and Proteins

A. Isolation of cDNA Encoding Rat and Mouse MDC Proteins

Knowledge of the human MDC gene sequence described herein was used asdescribed below to isolate and clone putative rat and mouse MDC cDNAs,which are intended as aspects of the invention.

To clone a rat MDC cDNA, a labelled probe was prepared using standardrandom primer extension techniques. A fragment of the human MDC cDNA wasgenerated by PCR, which fragment includes the MDC coding region plusapproximately 300 bases of 3′ untranslated sequence. This fragment waslabelled with ³²P-deoxyribonucleotides using the Random Primed DNALabeling idt (Boehringer Mannhein, Indianapolis, Ind.). The labelled MDCprobe was used to screen approximately 10⁶ bacteriophage lambda clonesfrom a commercially-available rat thymus cDNA library (Stratagene, LaJolla, Calif., Cat. No. 936502). Three positive clones were obtained.Sequencing of one of the positive clones, designated RT3, provided anapproximately 958 base pair sequence (SEQ ED NO: 37) that included anMDC open reading frame (SEQ ID NO: 38) and about 0.5 kb of 3′untranslated sequence. The open reading frame included sequence encodingthe putative mature MDC protein (SEQ ID NO: 38, residues 1 to 69) plus13 amino acids of the putative signal peptide sequence; it lacked theinitiator methionine codon and sequence encoding the amino terminus ofthe signal peptide. A complete rat MDC cDNA or genomic clone isobtainable using all or a portion of the RT3 sequence as a labelledprobe to re-probe the Stratagene rat cDNA library, and/or other rat cDNAlibraries, and/or a rat genomic DNA library.

To clone a mouse MDC cDNA, approximately 10⁶ bacteriophage lambda clonesof a commercially-available mouse thymus cDNA library (Stratagene, Cat.No. 935303) were screened with a radiolabeled fragment of theabove-described rat MDC cDNA. The probe was generated using overlappingprimers in a primer extension reaction. The primer extension reactioncomprised: partially overlapping primers corresponding to nucleotides 41to 164 of SEQ ID NO: 37 (and to nucleotides 92-215 of SEQ ID NO: 1);³²P-labelled deoxyribonucleotides; and the Klenow fragment of E. coliDNA polymerase. Twelve positive clones were isolated.

One positive clone, designated MT3, was sequenced and found to contain a1.8 kb cDNA insert that included the entire putative murine MDC codingregion and about 1507 bases of 3′ untranslated sequence. The cDNA anddeduced amino acid sequences for this murine MDC clone are set forth inSEQ ID NOs: 35 and 36, respectively. The mouse MDC has a putative 24amino acid signal sequence followed by a 68 amino acid MDC sequence.

Comparisons of the human, rat, and mouse MDC protein and DNA (codingregion) sequences reveal the following levels of similarity: Human vs.rat protein: 65% identity; Human vs. rat DNA: 74% identity; Human vs.mouse protein: 64% identity; Human vs. mouse DNA: 72% identity; Rat vs.mouse protein: 88% identity; Rat vs. mouse DNA: 92% identity.The four cysteines characteristic of C-C chemokines are conserved in allthree MDC proteins.

It is contemplated that the encoded rat and mouse MDC polypeptidescorresponding to SEQ ID NOs: 38 and 36 are processed into mature mouseMDC proteins, in a manner analogous to the processing of the human MDCprecursor, by cleavage of a signal peptide. The signal peptides for bothhuman and murine MDC are 24 amino acids. The exact length of the rat MDCsignal peptide will be readily apparent upon isolation of a full lengthrat MDC cDNA. It will be appreciated that these proteins can besynthesized recombinantly or synthetically and assayed for MDCbiological activities as described herein for human MDC. Likewise, itwill be appreciated that any analogs described herein for human MDC canbe similarly prepared for these other mammalian MDC proteins.

The foregoing results demonstrate the utility of polynucleotides of theinvention for identifying and isolating polynucleotides encoding othervertebrate MDC proteins, especially other mammalian or avian MDCproteins. Such identified and isolated polynucleotides, in turn, can beexpressed (using procedures similar to those described in precedingexamples) to produce recombinant polypeptides corresponding to othervertebrate forms of MDC, which proteins would be useful in the samemanners that human MDC is useful, including therapeutic veterinaryapplications. Polynucleotides encoding vertebrate (and especiallymammalian or avian) MDC proteins, the proteins themselves, and analogsthereof are all contemplated to be aspects of the present invention.

B. Synthesis of Murine MDC and Demonstration of Biological Activity

The interaction between murine MDC and human CCR4 was demonstrated usingsynthetic murine MDC in a chemotaxis assay. Murine L1.2 cellstransfected with human CCR4 (Example 25) were tested to determine ifsuch cells would migrate towards synthetic full-length mature murine MDC(SEQ ID NO: 36, residues 1 to 68) (Gryphon Sciences and IanClark-Lewis), and/or toward a synthetic murine MDC analog designated“Leu-MDC” which consists of a leucine residue attached to the aminoterminus of mature murine MDC. (Murine Leu-MDC is thus analogous to“MDC(n+1)” described in Example 11. Costar Transwells with 3 μm filterswere used for the assay.

Varying amounts of the synthetic MDC polypeptides (ten-fold dilutionsfrom 10000 to 1 ng/ml final concentrations) were added to 600 μlRPMI/0.5% BSA (endotoxin-free) in the lower wells and 10⁶ cells in 100μl RPMI/0.5% BSA (endotoxin-free) were added to the upper chambers.After incubating the transwells at 37° C. for 4 hours, the upperchambers-were transferred to 500 μl ice-cold PBS/0.5 mM EDTA to releaseany migrated cells still clinging to the underside of the filter. Cellswhich had migrated to the lower chambers were harvested by combining the600 μl medium from the lower chamber with the 500 μl PBS/EDTA for eachwell Cells were centrifuged, resuspended in 200 μl of 1% formaldehyde,and then counted for 30 seconds on the FACSCAN (Becton-Dickinson).

The number of L 1.2/huCCR4 cells that were observed to have migratedtoward full-length mature murine MDC showed a characteristicdose-response curve, with chemotaxis observed at 1 ng/ml MDC and withpeak chemotaxis occurring at 100 ng/ml murine MDC. The same number ofcells migrated towards the 100 ng/ml full-lenght mature murine MDC fromGryphon Sciences and Ian Clark-Lewis,—indicating that the twopreparations had equivalent activity. The responses of L1.2/huCCR4 cellsto murine Leu-MDC were approximately 20% lower than to full-length MDC.

C. Murine MDC Competes with Human MDC for Binding to Human CCR4.

In duplicate, 5×10⁵ L1.2/huCCR4 cells were incubated with 0.1 nM¹²⁵I-labeled human mature MDC, alone or with unlabeled human mature MDC(10 nM or 100 nM), murine mature MDC (100 nM), or the the chemokine LARC(100 nM, control), for one hour in 200 μl binding buffer (50 mM HEPES,pH 7.5, 1 mM CaCl₂, 5 mM MgCl₂, 0.5% BSA, and 0.05% azide). Cells werespun down at slow speed and washed twice with binding buffer plus 0.5 MNaCl. Fifty microliters of scintillant fluid was added and samples werecounted with a beta-counter. Unlabeled human and murine MDC bothsubstantially reduced the amount of labeled MDC that bound to theCCR4-expressing cells (approx. 4000 cpm versus less than 500 cpm), with100 nM murine MDC displaying a level of competition intermediate to thatof 10 nM and 100 nM human MDC. The contol chemokine LARC (whichspecifically binds CCR6) diplayed substantially no competitive bindingability (approx. 3800 cpm).

The foregoing assay results demonstrate that a nonhuman form of MDC(murine MDC) is capable of binding and stimulating cells expressing ahuman MDC receptor. This data demonstrates an indication for vertebrateMDC, MDC fragments and analogs, and MDC modulators for human treatmentsand treatment formulations, as described elsewhere herein for human MDC,MDC fragments and analogs, and MDC modulators.

D. Macaque MDC cDNA and Polypeptide Sequences.

Polymerase chain reaction (PCR), using oligonucleotides designed fromthe human MDC cDNA as primers, was performed in order to amplify andisolate a cDNA encoding macaque MDC from a macaque thymus cDNA library.The macaque MDC amino acid sequence with secretory signal sequence is 93amino acids and shares about 94% amino acid identity with human MDC.Referring to SEQ ID NO: 2, the macaque MDC amino acid sequence isidentical to that of the human sequence, with the following variations:valine at position-18; phenylalanine at position-17; glycine atposition-15; isoleucine at position-12; methionine at position 21; andserine at position 46. The macaque cDNA and deduced amino acid sequencesare set forth in SEQ ID NOs: 45 and 46.

E. Use of Multiple Vertebrate MDC Sequences to Design MDC Analogs

The amino acid sequences for human, macaque, mouse, rat and/or otheranimals can be aligned using any alignment algorithm known in the art.Such an alignment will identify positions and regions within the MDCsequences that are highly conserved (e.g., that are identical indifferent species), moderately conserved (e.g., identical in somespecies with substitutions in other species of amino acids of similarcharacter (e.g., acidic, basic, aliphatic, aromatic)), or variable(e.g., different in most or all species, including substitutions ofamino acids of different character). Such an alignment providessignificant guidance for the design of MDC analogs that will act as MDCmimetics as well as analogs that may act as MDC inhibitors. Substitutionor deletion of variable residues is more likely to result in analogsthat retain MDC biological activities, whereas highly conserved residuesare targets for alteration or deletion to design analogs havingdifferent activities or having MDC inhibitory activity.

EXAMPLE 29 Receptor Binding and Stimulation Assays

Using procedures essentially as described in Example 25, selected MDCanalogs described in Example 11 were screened for the ability to bindCCR4 and/or induce calcium (Ca⁺⁺ flux and chemotaxis in L1.2 cellstransfected with CCR4.

The analog MDC(n+1) bound CCR4 with similar affinity to MDC, but inducedcalcium flux and chemotaxis in L1.2/CCR4 cells with a slightly lowerpotency than MDC. For example, in chemotaxis, the peak activity forMDC(n+1) was observed at 100 ng/ml rather than 10 ng/ml, and the maximumnumber of cells migrating was 5000, compared to 9000 for MDC.

MDC(9-69) bound CCR4 with reduced affinity relative to that of MDC(0-69). MDC(9-69) did not induce calcium flux in L1.2/CCR4 cells, and itwas much less potent in chemotaxis. The fact that MDC(9-69) binds CCR4but does not signal through CCR4 indicates a utility of MDC(9-69) as anMDC inhibitor.

Collectively, the activities of MDC (n+1) and MDC (9-69) indicate thatamino-terminal additions and deletions and other modifications mayresult in useful MDC inhibitors.

The analog “MDC-wvas” bound CCR4 with ˜500-fold less affinity than MDC,induced only a very small calcium flux, and did not induce anychemotaxis. The analog “MDC-eyfy” acted similar to MDC in CCR4-binding,chemotaxis, and calcium flux assays.

EXAMPLE 30 Monoclonal Antibodies 252Y & 252Z Inhibit CCR4-MediatedCellular Responses to MDC

Using procedures similar to those described in Example 25, themonoclonal antibodies 252Y and 252Z described in Example 18 werescreened for the ability to modulate MDC-CCR4 binding and modulate theCCR4-mediated biological activities of MDC.

A. Antibodies 252Y and 252Z Inhibit MDC Binding to CCR4

The fusion protein MDC-SEAP (Example 25) was employed to evaluate theability of the antibodies to inhibit MDC binding to its receptor CCR4.MDC-SEAP at a concentration of 0.5 nM was incubated for fifteen minutesat room temperature with varying concentrations (0.01-10 μg/ml, shown inFIG. 11) of antibody 252Y, antibody 252Z, or an isotype control (finalreaction volume 100 μl). Thereafter, the mixtures were added toCCR4-expressing L1.2 cells (100 μl, 4000 cells per μl), and incubated at4° C. for an additional 60 minutes. The extent of MDC-SEAP binding tothe CCR4-expressing cells was determined by alkaline phosphatasechemiluminescent assay as described in Example 25. A baseline level ofnon-specific binding (defined as the amount of binding that could not becompeted by a 200-fold molar excess of native MDC) was determined andsubtracted from experimental measurements. FIG. 11 presents theexperimental results in graphical form, wherein each data pointrepresents a percentage of maximum binding. (Maximum binding was definedas the amount of MDC-SEAP bound to the cells in the absence of antibody,minus non-specific binding.) As shown in FIG. 11, both antibody 252Y andantibody 252Z (but not the isotype control) inhibited MDC-SEAP bindingto CCR4-infected cells in a dose-dependent manner. Fifty percentinhibition of binding was observed for both antibodies at an antibodyconcentration of about 2 μg/ml.

B. Antibodies 252Y and 252Z Inhibit MDC-Induced Chemotaxis

To confirm that antibodies 252Y and 252Z also were capable of inhibitingCCR4-mediated cellular responses to MDC, both calcium flux andchemotaxis assays were performed using the CCR4-transfected L1.2 cells.

For the calcium flux assay, the transfected L1.2 cells were labelledwith Fura-2/AM (see Example 19) and monitored for Ca⁺⁺-inducedfluorescence changes using an ANCO-Bowman Series 2 fluorimeter. Additionof 75 nM MDC to the cells induced a rapid, transient increase inintracellular Ca⁺⁺ levels. This Ca⁺⁺ flux response was completelyinhibited when either antibody 252Y or antibody 252Z were added to thecells at a concentration of 10 μg/ml one minute before contacting thecells with the MDC solution. An isotype-matched control antibody had noeffect on the MDC-induced Ca⁺⁺ flux. Thus, both antibodies blocked thecalcium flux response to MDC in CCR4-transfected L1.2 cells.

For the chemotaxis assay, CCR4-transfected L1.2 cells (approx. 10million cells/ml in a volume of 0.1 ml) were preincubated with antibody252Y, antibody 252Z, or an isotype-matched control in RPMI-1640 media(Gibco) at various concentrations ranging from 0.5 to 50 μg/ml for 30minutes at room temperature. Thereafter, the cells were exposed to 100ng/ml MDC (i.e., the peak concentration for maximum chemotaxis) for 4hours in a Costar Transwell apparatus. The number of cells migratingtoward MDC was counted using a Becton-Dickinson FACScan apparatus. Asshown in FIG. 12, MDC-induced chemotaxis of these cells was totallyinhibited by either antibody 252Y or antibody 252Z at concentrations of2-5 μg/ml, but not by the isotype-matched control. The IC₅₀ antibodyconcentration (required to inhibit 50% migration) was 1 μg/ml. The sameantibodies did not inhibit chemotaxis of the CCR4/L1.2 cells toward theC-C chemokine TARC, indicating that the inhibitory effect was specificfor MDC.

In a similar set of experiments, antibody 272D was screened for itsability to inhibit MDC stimulated chemotaxis. Ten μg/ml of antibody 272Dwas required to inhibit chemotaxis toward recombinant MDC (30 ng/ml) bygreater than 90%. Only 2 μg/ml of antibody 252Z was required to achievea similar level of inhibition, indicating that antibody 252Z is a morepotent inhibitor of MDC induced chemotaxis.

EXAMPLE 31 MDC Induces Chemotaxis of T_(H)2 Helper T Cells

A transendothelial migration assay was performed essentially asdescribed in the art [Ponath, et al., J. Clin. Invest., 97: 604-612(1996); Ponath et al., J. Exp. Med., 183: 2437-2448 (1996); and Imai, etal., Cell, 91:521-530 (1997)] to determine the presence and thephenotype of T cells that migrate toward the chemokines TARC and MDC.Briefly, about 2×10⁵ cells of the endothelial cell line ECV304 (ATCCCRL-1998 or European Cell Culture Collection, Portions Down, UTK) wereadded to Transwell inserts (Coaster) with a 5 μm pore size and culturedat 37° C. for 48-96 hours in M199 medium (GIBCO/BRL) supplemented with10% FCS. Chemokines were diluted (serial dilutions of 0.1 to 100 nM) ina migration medium (a 1:1 mixture of RPMI-1640:M199, supplemented with0.5% BSA, 20 mM HEPES, pH 7.4) and added to 24-well tissue cultureplates in a final volume of 600 μl. Endothelial cell-coated inserts wereplaced in each well and 10⁶ peripheral blood mononuclear cells (PBMC) orT cell lines in 100 μl were added to the upper chambers. The cells wereallowed to migrate through the endothelial cells into the lower chambersat 37° C. for 4 hours (PBMC) or 90 minutes (T cell lines). The migratedcells in the lower chambers were stained with FITC- or PE-conjugatedmonoclonal antibodies (mAb) for indicated cell surface makers andcounted by flow cytometry.

In the transendothelial cell migration assay, both TARC and MDC induceddose-dependent vigorous migration of CD14⁻ lymphocytes but not of CD14⁺monocytes, with MDC consistently inducing cell migration about 2 timesmore efficiently than TARC. Migration activity was detected withchemokine concentrations as low as 1 nM. Significant migration occurredwith 10 nM TARC and 10 nM MDC. Analysis of the migrating lymphocytesrevealed that 10 nM of either TARC or MDC attracted predominantly CD4³⁰T cells. Neither TARC nor MDC induced migration of CD19⁺ B cells orCD16⁺ NK cells. Furthermore, TARC and MDC attracted almost exclusivelyCD45RA⁻/CD45RO⁺ effector/memory T cells. This observation was consistentwith the observation that a murine (IgG) monoclonal antibody to CCR4stained highly selectively a fraction (˜20%) of CD45RO⁺CD4⁺ memoryhelper T cells.

Effector/memory helper T cells represent a population of cells that haveencountered cognate antigens in vivo and have differentiated into T_(H)1or T_(H)2 cells. Since CCR4 is expressed on about 20% of effector/memoryhelper T cells, additional experiments were conducted to determinewhether CCR4 is selectively expressed on certain subsets of helper Tcells.

First, CD4⁺CD45RO⁺ T cells (obtained from PBMC by negative selectionwith Dynabeads (Dynal) after incubation with anti-CD16, anti-CD14,anti-CD20, anti-CD8, and anti-CD45RA antibodies) were fractionated intoCCR4⁺ and CCR4⁻ subpopulations by staining with the anti-CCR4 mAb andcell sorting. The cell subpopulations were expanded as polyclonal celllines by culturing for 9-14 days at 37° C. in RPMI medium supplementedwith PHA (diluted 1:100) and 100 U/ml IL-2. Expanded cells weresubjected to a second round of enrichment by staining with anti-CCR4monoclonal antibody and sorting. Sorted cells were immediately activatedwith 50 ng/ml PMA (Sigma) and 1000 ng/ml ionomycin (Sigma) for 24 hours,at which time the culture medium was analyzed by ELISA (R&D) todetermine each population's pattern of cytokine production. Since helperT cells are classified into T_(H)1 and T_(H)2 subsets based on theirprofiles of cytokine production [Mosmann et al., Immunol. Today, 17:138-146 (1996)], this analysis permitted determination of whether CCR4is selectively expressed in one or the other subpopulation.

Analysis of the culture medium revealed that the CCR4⁺ T cells producedsignificantly larger amounts of IL-4 and IL-5 than the cultured CCR4⁻ Tcells (>12 ng/ml for CCR4⁺ T cells versus<2.5 ng/ml for CCR4⁻ T cellsfor each cytokine). Conversely, CCR4⁻ T cells produced IFN-γ at levelsmuch higher than CCR4+T cells (>300 ng/mnl vs.<0.25 ng/ml). Thesecytokine expression patterns indicate that the CCR4+population of cellscontained almost exclusively T_(H)2 cells, whereas CCR4⁻ cells wereenriched for T_(H)1 cells.

To support the conclusion that CCR4⁺ T cells are predominantly T_(H)2cells, the CD4⁺ CD45RO⁺ T cells that had been attracted by TARC or MDCin the transendothelial migration assay were expanded by culturing inPHA and IL-2 and then examined for their pattern of cytokine productionas described above. Compared to total CD4⁺CD45RO⁺ T cells, the cellsattracted by TARC or MDC were enriched for producers of IL-4 and IL-5and depleted of producers of IFN-γ.

To further confirm the observed selective expression of CCR4 on T_(H)2cells, experiments were performed to polarize CD4⁺CD45RA⁺ naive T cellsin vitro, and the artificially polarized cell populations were examinedfor CCR4 expression. The naive T cells (obtained from PBMC by negativeselection with Dynabeads after incubation with anti-CD16, anti-CD 14,anti-CD20, anti-CD8, and anti-CD45RO antibodies) were polarized intoT_(H)1 cells by culturing in the presence of PHA (1:100), 2 ng/ml IL-12,and 200 ng/ml anti-IL-4 monoclonal antibodies (Pharmingen); or intoT_(H)2 cells by culturing with PHA (1:100), 10 ng/ml IL-4, and 2 μg/mlanti-IL-12 monoclonal antibodies. After 3-4 days, 100 U/ml IL-2 wasadded to the cultures. CCR4 expression and transmigration were analyzedat day 9-14.

Analysis of the cultured cells with an anti-CCR4 monoclonal antibodyrevealed that 60% of cells polarized into T_(H)2 cells expressed CCR4,compared to only 4% of cells polarized into T_(H)1 cells. Northern blotanalysis of the RNA isolated from these cell populations alsodemonstrated that T_(H)2 cells expressed CCR4 mRNA at levels much higherthan T_(H)1 cells. As controls, CCR7 mRNA was expressed in both types ofcells whereas CCR3 mRNA was not detected in either type of cell.

In the endothelial transmigration assay, the artificially polarizedT_(H)2 cells, but not those polarized into T_(H)1, migrated vigorouslytoward TARC and MDC, whereas both types of cells migrated toward SLC.(See Nagira, et al., “Molecular cloning of a novel human CC chemokinesecondary lymphoid-tissue chemokine that is a potent chemoattractant forlymphocytes and mapped to chromosome 9p13,” J. Biol. Chem., 272:19518-19524 (1997).) Neither population of cells migrated towardeotaxin, a ligand for CCR3.

Collectively, the foregoing experiments demonstrate that a significantpopulation of T_(H)2 cells express the chemokine receptor CCR4, and thatthe chemokines TARC and MDC represent selective chemoattractants ofT_(H)2 cells, an effect that presumably is mediated at least in partthrough CCR4. Tissues of allergic inflammation are infiltrated by T_(H)2cells, as well as by eosinophils, another cell type selectivelyattracted by MDC (see Example 12). Furthermore, T cells migrating intotissues after antigen challenge have been reported to be involved inlocalized production of the T_(H)2 cytokines, IL-4 and IL-5, and inaccumulation of eosinophils. (See Garlisi et al., Clin. Immunol.Immunopathol., 75: 75-83 (1995).) Additionally, TARC and MDC areabundantly produced by dendritic cells whose close interactions withmigrating lymphocytes constitute essential parts in initiation andpromotion of immune responses. (See Steinman, R. M., Annu. Rev.Immunol., 9: 271-296 (1991).) Enhanced TARC and MDC production fromantigen presenting cells in T_(H)2 responses would be expected to leadto further recruitment of T_(H)2 cells via CCR4. Thus, the discoveriesherein relating to the biological effects of MDC indicate that theeffects may be deeply intertwined and involved in multiple aspects of animmunological or allergic cascade, a factor of direct clinicalimportance. For example, agents that interfere with the interactions ofTARC or MDC with the receptor CCR4 (and/or that interfere with theinteractions of TARC or MDC with T_(H)2 cells or eosinophils incell-based assays) have therapeutic indications for reducing allergicinflammatory responses. The use of such agents in the treatment ofasthma, a conditions characterized by eosinophilic infiltration andprobable involvement of presentation of sensitizing antigen by mucosaldendritic cells to T_(H)2 T cells, is specifically contemplated.

EXAMPLE 32 Use of MDC and MDC Antagonists to Modulate PlateletAggregation

The following experimental data indicates that MDC promotes plateletaggregation, and suggests a therapeutic indication for MDC and MDCantagonists to modulate platelet aggregation.

Female Lewis rats, six to eight weeks old, were administered 0.5 μg ofsynthetic mature human MDC(1-69) intravenously in a saline solution, viathe tail vein. At various time points, the animals (4) were anesthetizedwith 100 μl ACE cocktail (Ketamine, ACE promazine and Rompon) and bloodsamples were collected into Microcontainers containing EDTA (BecktonDickinson). Samples (300-400 μl) were stored overnight at 4-8° C. A CBCwith Differential analysis was conducted to identify changes in cellnumber in the rats compared to control rats that had been administeredonly phosphate-buffered saline. In all four animals treated, markedplatelet aggregation was observed. This aggregation was most pronouncedat the time of MDC administration and dissipated with time after thebolus. A similar phenomenon was observed in mice using an analogousprotocol.

Receptor analyses have indicated that platelets express detectablelevels of the MDC receptor CCR4. These experiments suggest a receptorthrough-which MDC may exert its platelet-aggregating effects.

The foregoing observations suggest that mature MDC stimulates plateletaggregation, and suggests that MDC antagonists are useful for inhibitingcoagulation. Such use is indicated, e.g., in myocardial infarctionpatients to prevent further inappropriate blood clotting, and inpatients for the therapeutic or prophylactic treatment of stroke.

The concentrations at which MDC induces platelet aggregation and atwhich MDC antagonists prevent platelet aggregation are determined invitro using purified platelets and serial dilutions of MDC and MDCantagonists and procedures that are well known in the art. See, e.g.,Jeske et al., Thromb. Res., 88(3):271-281 (1997); Herault et al.,Thromb. Haemost., 79(2):383-388 (1998); and Furakawa et al., Jpn. J.Pharmacol., 75(3):295-298 (1997). Putative MDC antagonists for screeningin such assays include all of the putative MDC antagonists identifiedabove, e.g., in Example 20. Those MDC analogs that inhibit plateletaggregation and those that promote aggregation are determined by suchdose response studies and/or by mouse studies as described above.

Similarly, since TARC also signals through CCR4, the use of TARC andTARC antagonists to modulate platelet aggregation also is intended as anaspect of the invention.

EXAMPLE 33 Use of an MDC Antagonist to Modulate an Immune Response in aMammalian Host

The following procedures are performed to demonstrate that MDCantagonists, such as MDC neutralizing antibodies, are capable ofmodulating an immune respone in a mammalian host.

A. Antigen-Induced Asthma Model

Laboratory animals (e.g., Balb/C mice) are challenged with ovalbuminusing the following regimen: Day 0: 100 μg ovalbumin (Sigma), 4.5 mgalum (Imject®, Pierce), administered by 200 μl intraperitonealinjection; Day 14: 100 μg ovalbumin, 4.5 mg alum administered by 200 μlintraperitoneal injection, plus 100 μg ovalbumin in 50 μl saline,administered intra-nasally; days 25, 26, and 27: 50 μg ovalbumin in 50μl saline, administered intra-nasally. As a contol, saline isadministered to animals in lieu of ovalbumin. To test the effect of aputative MDC modulator (such as an MDC-neutralizing antibody) on theanimal's allergic-type response to the ovalbumen, the modulator (or acontrol, e.g., saline) is administered to test animals intraperitoneallyon days 25, 26, and 27, one hour prior to challenge with ovalbumin.Exemplary dosing of an anti-MDC antibody is 0.1 to 5 mg/kg body weight.

On day 28, the mice are sacrificed, blood is collected, andbronchioalveolar lavage is performed. Cells from the lavage fluid arecollected and counted, and a white blood cell differential is performed.Reduction in eosinophils and/or neutrophils in the lavage fluid oftreated animal versus contol animals is indicative of the therapeuticefficacy of the MDC antagonist treatment. Reduction in anti-ovalbumenantibodies (especially IgE antibodies) in the blood (assayed by ELISA,for example) is further indicative of the therapeutic efficacy of theMDC antagonist.

B. Modulation of a T_(H)2 Response

To demonstrate the ability of an MDC antagonist to suppress an immuneresponse, laboratory animals are immunized subcutaneously orintraperitoneally with a suitable antigen, such as ovalbumin or tetanustoxoid, or with a saline control. Aluminum hydroxide (alum), whichpreferentially promotes a T_(H)2 response, or Freund's completeadjuvant, which tends to drive a T_(H)1 response, are used as adjuvantsin some of the animals. Animals are immunized on day 0 (e.g., with 100μg ovalbumin+4.5 mg alum), followed by booster immunizations at, e.g.,days 14 and 28. The antibody titer against the selected antigen ispermitted to drop to normal levels in the animals, e.g., for 1-2 months,monitored via ELISA.

After antibody levels have dropped to normal, the animals arere-challenged with the selected antigen. An MDC antagonist, such as a anMDC-neutralizing antibody, is administered contemporaneously with theantigen, two, six, and/or twenty-four hours later. One week later, bloodfrom the animals is drawn, white blood cells are analyzed, andantibodies to the antigen are titered and isotyped. Reduced levels ofIgG₁ antibody, IgE antibody, and T_(H)2 cells in the treated animalsversus the control animals is indicative of a therapeutically effectiveMDC antagonist, where immunosuppression is desired. A more pronouncedthereapeutic effect in the alum-administered animals than the animalsinjected with Freund's aduvant is expected.

C. Murine Lupus Model

The therapeutic efficacy of an MDC antagonist for the treatment of lupuserythematosus is demonstrated in animal models, such as NZB/NZW F1 mice,that are known in the art and have been described in the literature.See, e.g., Wofsy, D. et al., J. Immunol., 138(10): 3247-3253 (May,1987); and Daikh et al., J. Immunol., 159(7): 3104-3108 (October 1997).

D. Use of an MDC Antagonist to Treat Human Lupus Erythematosus

An MDC antagonist such as a humanized or human anti-MDC antibody oranti-CCR4 antibody is employed in a standard dose-escalation study todemonstrate efficacy in the treatment of lupus erythematosus in affectedhuman individuals. Exemplary dosing regimens for an antibody range from0.01 to 50 mg/kg body weight, and preferably 0.1 to 5 mg/kg,administered weekly, or bi-weekly, or monthly. Treatment efficacy isdetermined by monitoring standard indices. See, e.g., Bombardier et al.,“Derivation of the SLEDAI: a disease activity index for lupus patients,”Arthritis Rheum, 35: 630-640 (1992); Liang et al., “Measurement ofsystemic lupus erythematosus activity in clinical research,” ArthritisRheum., 31: 817-825 (1988). Optimal dosing is determined by standarddose-response studies after efficacy is demonstrated.

E. Use of an MDC Antagonist to Treat Human Multiple Sclerosis

An MDC antagonist such as a humanized or human anti-MDC antibody oranti-CCR4 antibody is employed in a standard dose-escalation study todemonstrate efficacy in the is treatment of multiple sclerosis inaffected human individuals. Exemplary dosing regimens for anantibody-based therapeutic are as set forth in Seciton D, above.Treatment efficacy is determined by monitoring standard MS indices. See,e.g., Kurtzke, J. F., “Rating neurologic impairment in multiplesclerosis: An expanded disability status scale (EDSS),” Neurology, 33:1444 (1983).

The biological functions of MDC, elucidated as described above, suggestseveral clinical applications.

Chemokines in general attract and activate monocytes and macrophages(Baggiolini et al., supra), and MDC in particular attracts macrophagesand inhibits monocyte chemotaxis. Thus, MDC expression in a pathogenicinflammatory setting may exacerbate disease states by recruitingadditional macrophages or other leukocytes to the disease site, byactivating the leukocytes that are already there, or by inducingleukocytes to remain at the site. Thus, inhibiting the chemoattractantactivity of MDC may be expected to alleviate deleterious inflammatoryprocesses. Significantly, the potential benefits of such an approachhave been directly demonstrated in experiments involving IL-8, a C-X-Cchemokine that attracts and activates neutrophils. Antibodies directedagainst IL-8 have a profound ability to inhibit inflammatory diseasemediated by neutrophils [Harada et al., J. Leukoc. Biol., 56:559(1994)]. Inhibition of MDC is expected to have a similar effect indiseases in which macrophages are presumed to play a role, e.g., Crohn'sdisease, rheumatoid arthritis, or atherosclerosis.

Alternatively, augmenting the effect of MDC may have a beneficial rolein such diseases, as chemokines have also been shown to have a positiveeffect in wound healing and angiogenesis. Thus, exogenous MDC or MDCagonists may be beneficial in promoting recovery from such diseases.

In addition, the myelosuppressive effect demonstrated for the C-Cchemokine MIP-1α (Maze et al., supra) suggests that MDC may have asimilar activity. Such activity, provided by MDC or MDC agonists, mayyield substantial benefits for patients receiving chemotherapy orradiation therapy, reducing the deleterious effects of the therapy onthe patient's myeloid progenitor cells.

MDC or MDC agonists may also prove to be clinically important in thetreatment of tumors, as suggested by the ability of the C-C chemokineTCA3 to inhibit tumor formation in mice (see Laning et al., supra). MDCmay act directly or indirectly to inhibit tumor formation, e.g., byattracting and activating various non-specific effector cells to thetumor site or by stimulating a specific anti-tumor immunity. Thefibroblast-antiproliferative effect of MDC indicates a therapeuticutility for MDC in the treatment of diseases such as pulmonary fibrosisand tumors, in which enhanced or uncontrolled cell proliferation is amajor feature.

While the present invention has been described in terms of specificembodiments, it is understood that variations and modifications willoccur to those skilled in the art. Accordingly, only such limitations asappear in the appended claims should be placed on the invention.

1. A purified polypeptide selected from the group consisting of: (a)non-human vertebrate Macrophage Derived Chemokine (MDC) polypeptides;(b) fragments of said non-human, vertebrate MDC polypeptides that retainat least one biological activity of the MDC polypeptide; and (c)fragments of said non-human vertebrate MDC polypeptides that are capableof inhibiting at least one biological activity of the MDC polypeptide.2. A purified polypeptide according to claim 1 that is a non-humanvertebrate MDC polypeptide or fragment thereof that retains at least onebiological activity of the vertebrate MDC polypeptide.
 3. A purifiedpolypeptide according to claim 1 that is a fragment of a non-humanvertebrate MDC polypeptide, said fragment being capable of inhibiting atleast one biological activity of the MDC polypeptide.
 4. A purifiedpolypeptide according to any of claims 1-3, selected from the groupconsisting of: (a) a polypeptide comprising a sequence of amino acidsidentified by positions 1 to 68 of SEQ ID NO: 36; (b) a polypeptidecomprising a sequence of amino acids identified by positions 1 to 69 ofSEQ ID NO: 38; and (c) a polypeptide comprising a sequence of aminoacids identified by positions 1 to 69 of SEQ ID NO:
 46. 5. Apharmaceutical composition comprising a purified polypeptide accordingto any one of claims 1-4 in a pharmaceutically acceptable carrier.
 6. Apurified polynucleotide comprising a nucleotide sequence that encodes apolypeptide according to any one of claims 1-4.
 7. A vector comprising apolynucleotide according to claim
 6. 8. A host cell stably transformedor transfected with a polynucleotide according to claim 6, or with avector comprising, said polynucleotide, in a manner allowing theexpression in said host cell of the polypeptide encoded by saidpolynucleotide.
 9. A method for producing a polypeptide that is anon-human vertebrate MDC or MDC fragment or analog, said methodcomprising growing a host cell according to claim 8 in a nutrient mediumand isolating the polypeptide from said cell or said medium.
 10. Anantibody that specifically binds to an MDC polypeptide, said antibodyselected from the group consisting of antibody 252Y and antibody 252Z.11. A hybridoma cell line that produces an antibody according to claim10.
 12. A kit for assaying for MDC polypeptides, said kit comprising, inassociation, two monoclonal antibodies that specifically bind MDC,wherein at least one of said monoclonal antibodies is a monoclonalantibody according to claim
 10. 13. A method for identifying a modulatorof binding between Macrophage Derived Chemokine (MDC) and an MDCreceptor, comprising the steps of: a) contacting an MDC receptorcomposition and a vertebrate Macrophage Derived Chemokine (MDC)polypeptide or fragment or analog thereof that binds chemokine-receptorCCR4, in the presence and in the absence of a putative modulatorcompound, wherein said receptor composition comprises cell membranes ofcells recombinantly modified to express increased amounts of thechemokine receptor CCR4; b) detecting binding between the receptorcomposition and the polypeptide; and c) identifying a putative modulatorcompound in view of decreased or increased binding between the receptorcomposition and the polypeptide in the presence of the putativemodulator, as compared to binding in the absence of the putativemodulator.
 14. A method for identifying a modulator of binding betweenMacrophage Derived Chemokine (MDC) and an MDC receptor, comprising thesteps of: a) contacting an MDC receptor composition and a vertebrateMacrophage Derived Chemokine (MDC) polypeptide in the presence and inthe absence of a putative modulator compound, wherein said receptorcomposition comprises eosinophil cell membranes; b) detecting bindingbetween the receptor composition and the polypeptide; and c) identifyinga putative modulator compound in view of decreased or increased bindingbetween the receptor composition and the polypeptide in the presence ofthe putative modulator, as compared to binding in the absence of theputative modulator.
 15. A method according to claim 13 or 14 wherein thepolypeptide is a vertebrate MDC polypeptide.
 16. A method according toclaim any one of claims 13-15, wherein said contacting step comprisescontacting said cell membranes with said polypeptide, and wherein saidmethod further comprises steps of recovering said cell membranes aftersaid contacting step; and washing said cell membranes prior to saiddetecting step to remove unbound polypeptide.
 17. A method according toany one of claims 13-16 wherein said polypeptide comprises a detectablelabel, and wherein in step (b) binding between the receptor compositionand the polypeptide is detected by detecting labeled polypeptide boundto the receptor composition.
 18. A method according to any one of claims13-16, wherein the receptor composition comprises a whole cellexpressing an MDC receptor on its surface, and wherein, in step (b),binding between the receptor composition and the polypeptide is detectedby measuring a binding-induced physiological change in said cell.
 19. Amethod according claim 18 wherein the binding-induced physiologicalchange is selected from the group consisting of: (a) the conversion ofGTP to GDP in said host cell; and (b) a change in the concentration ofcAMP in said host cell.
 20. A purified compound that is a modulator ofbinding between the chemokine MDC and an MDC receptor, said compoundidentified by a method according to any of claims 13-19.
 21. The use ofan MDC antagonist or TARC antagonist compound for preparation of amedicament for inhibiting platelet aggregation in a mammalian subject.22. The use of an MDC antagonist or TARC antagonist compound forpreparation of a medicament for the treatment or palliation of lupuscrythematosus in a mammalian subject.
 23. The use of an MDC antagonistcompound for preparation of a medicament for inhibiting MDC-inducedactivation, chemotaxis, or proliferation of cells that express thechemokine receptor CCR4.
 24. The use of an MDC antagonist or TARCantagonist compound for preparation of a medicament for inhibiting anallergic reaction in a mammalian host.
 25. The use of an MDC antagonistor TARC antagonist compound for preparation of a medicament for thetreatment of asthma.
 26. A method of palliating an allergic reaction ina mammalian subject, comprising the steps of: identifying a mammaliansubject in need of treatment for an allergic reaction that ischaracterized by eosinophil accumulation, and administering to saidmammalian subject a composition comprising an MDC antagonist compound orTARC antagonist compound in an amount effective to palliate the allergicreaction.
 27. A method of treating a disease state characterized byaggregation of platelets in a mammalian subject, comprising the stepsof: identifying a mammalian subject in need of treatment for saiddisease state, and administering to said mammalian subject a compositioncomprising an MDC antagonist compound or TARC antagonist compound in anamount effective to prevent platelet aggregation in said mammaliansubject.
 28. A method of treating lupus crythematosus in a mammaliansubject, comprising the steps of: identifying a mammalian subject inneed of treatment for lupus erythematosus, and administering to saidmammalian subject a composition comprising an MDC antagonist compound orTARC antagonist compound in an amount effective to treat lupuscrythematosus or palliate its symptoms.
 29. A method of treating adisease state characterized by activation, chemotaxis, or proliferationof cells that express the chemokine receptor CCR4 in a mammaliansubject, comprising the steps of: identifying a mammalian subject inneed of treatment for said disease state, and administering to saidmammalian subject a composition comprising an MDC antagonist compound orTARC antagonist compound in an amount effective to prevent at least oneof activation, chemotaxis, and proliferation of cells that express thechemokine receptor CCR4 in said mammalian subject.
 30. A use accordingto any of claims 21-25 or a method according to any of claims 26-29wherein the MDC antagonist compound is selected from the groupconsisting of: (a) a polypeptide fragment or analog of a vertebrate MDCthat inhibits MDC activation of an MDC receptor, (b) an antibody thatspecifically binds a vertebrate MDC polypeptide; (c) an MDC antagonistaccording to claim 20; (d) a polypeptide capable of binding to avertebrate MDC polypeptide and comprising an antigen-binding fragment ofan anti-MDC antibody; (c) a polypeptide comprising the C-C chemokinereceptor 4 (CCR4) amino acid sequence set forth in SEQ ID NO: 34 orcomprising a continuous fragment thereof that is capable of binding toMDC; and (f) combinations of (a)-(e).
 31. A use according to any ofclaims 21-25 or a method according to any of claims 26-29 wherein saidMDC antagonist compound comprises an antibody substance that binds MDC,said antibody substance selected from the group consisting of monoclonalantibodies, polyclonal antibodies, single chain antibodies, chimeric,antibodies, and humanized antibodies.
 32. In a vaccine composition, theimprovement wherein a polypeptide is included in the vaccinecomposition, said polypeptide comprising a vertebrate MDC polypeptide orbiologically active fragment or analog thereof.
 33. A method ofstimulating an immune response in a human or animal comprising the stepof administering a vaccine composition according to claim 32 to a humanor animal in an amount effective to stimulate an immune response in thehuman or animal.
 34. A method of screening a patient suspected ofsuffering from, or undergoing treatment for, a disorder characterized byMDC-induced T_(H)2 cell migration or activation, comprising the stepsof: obtaining a fluid sample from a patient suspected of suffering froma disorder characterized by MDC-induced T_(H)2 cell migration oractivation; and determining the concentration of MDC in the fluidsample.
 35. A method according to claim 34, wherein the fluid comprisesserum, and wherein the MDC concentration is determined via ELISA assay.36. A method according to claim 34, wherein the patient is suspected ofsuffering from the disease state, and wherein an elevated MDCconcentration is considered diagnostic of the disease state.
 37. Amethod according to claim 34, wherein the patient is undergoingtreatment for the disease state, and MDC concentration in the fluidsample is used to monitor dosing or efficacy of treatment.